Gut-Modulating Agents and Amyotrophic Lateral Sclerosis: Current Evidence and Future Perspectives

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a rare but highly fatal neurodegenerative disorder characterized by the progressive loss and degeneration of motor neurons in the brain and spinal cord. Its average age of onset is currently estimated at 61.8 years, with death typically occurring 3–5 years following diagnosis [1,2]. Although the incidence of ALS is around 2.0 cases per 100,000 persons per year, its global prevalence is closer to 5.4 cases per 100,000 persons, reflecting a low survival rate [2]. The clinical manifestations of ALS are heterogeneous and can be initially non-specific. Patients afflicted with the disease often experience debilitating muscle weakness, stiffness, or paralysis, which begin in the extremities and then spread to involve most parts of the body. Despite extensive research efforts, the etiology of ALS remains largely unknown, and thus effective treatment options that can delay disease progression are limited. However, a growing body of evidence has recently implicated enteric microbiota and the gut–brain axis (GBA), an intricate two-way communication system between the gastrointestinal (GI) tract and central nervous system (CNS), in the pathogenesis of several neurodegenerative diseases, including ALS [3].

Factors contributing to overall gut health, such as intestinal inflammation and barrier permeability, can have profound effects on brain function and well-being [3,4]. Symptoms of GI upset or dysfunction (e.g., pain, dysphagia, reflux, and constipation) have also been noted in ALS, further suggesting a link between gut and brain pathology [5,6,7]. Moreover, imbalances or alterations in the composition of the gut microbiome, commonly referred to as gut dysbiosis, have been observed in human patients as well as in experimental animal models of ALS [8,9]. Although several advancements in our understanding of the gut microbiome and its relation to neurodegenerative disorders have been made, the field is still in its early stages. Further explorations into the complex role of the GBA and its contributions to brain development in both health and disease can provide new insights into the mechanisms underlying ALS pathogenesis. In this review, we comprehensively summarize and present the current literature on gut dysbiosis in ALS, with a specific focus on the involvement of GBA pathology. Moreover, we aim to explore the potential of direct gut modulation as a therapeutic measure in the management of this devastating motor disease.

Bidirectional communication between the gut and brain is primarily mediated by the nervous, immune, and endocrine pathways [10,11]. Nervous pathways include the enteric nervous system (ENS), autonomic nervous system (ANS), and vagus nerve. Visceral afferents normally transmit sensory information from the gut to the CNS, which, in turn, modulates GI function by efferent signals. Intestinal motility, absorption, secretion, and blood flow are all regulated by the ANS [11]. Interestingly, the ANS shares several mediators and receptors with the gut’s immune system and, thus, can also play a role in regulating local inflammatory processes [12]. Signaling in the immune pathways is primarily mediated by enterocytes, resident immune cells, and the gut microbiota. Enterocytes, particularly a specialized subset called tuft cells, and gut-associated immune cells, such as macrophages, neutrophils, and dendritic cells, all express toll-like receptors (TLRs) that can recognize molecular patterns on the surfaces of invading pathogens and initiate an innate immune response [11]. Upon activation, these cells release inflammatory cytokines and chemokines that not only aid in further immune cell recruitment but also trigger signaling cascades that can communicate with the CNS. Cytokines may also act locally on vagal afferents [10] and subsequently affect signaling within the GBA. Endocrine pathways mainly involve hormones and the hypothalamic–pituitary–adrenal (HPA) axis. However, neuroactive microbial products, such as short-chain fatty acids (SCFAs) and secondary bile acids (2-BAs), can also propagate signals to the CNS either directly, by accessing the body’s systemic circulation, or indirectly, by interacting with enteroendocrine, enterochromaffin, and immune cells of the gut [10]. While this section offers a simplified overview of these pathways, it is important to recognize how different components of the GBA are related and work in concert to maintain gut homeostasis. Furthermore, it becomes evident that disruption in any component of the GBA can not only impact the gut but may also have downstream effects on the CNS.

The human gut is habitat to a highly complex yet balanced network of commensal microorganisms spanning all three domains of life: bacteria, archaea, and eukarya (including fungi, yeasts, and protozoa) [13]. Bacteriophages and eukaryotic viruses are also integral components of the gut microbiome [14]. Although the exact composition and diversity of microbial communities throughout the GI tract vary considerably, the gut microbiome is primarily dominated by bacterial populations. Specifically, among the eight identified bacterial phyla in the human gut, Firmicutes and Bacteroidetes constitute the majority portion and represent over 90% of all intestinal microbiota [13,15]. The remaining 10% is typically composed of Actinobacteria and smaller proportions of Proteobacteria, Verrucomicrobia, or Cyanobacteria [13,15,16]. The division of the intestinal flora at this taxonomic level is similar, if not uniform, across most healthy humans. However, each individual possesses a unique microbiome made up of different species and strains at varying densities likely owing to a number of genetic factors and host–microbe interactions [17,18]. Age, diet, lifestyle, environment, and disease may also account for individualized differences since they can shape and alter the gut’s microbial composition [15]. Furthermore, studies have shown that dietary changes or acute lifestyle modifications can quickly and reproducibly alter the gut microbiota [19,20,21]. Over the past decade, the relationship between gut health and neurological disorders has become a subject of research interest. Preclinical evidence implicates gut dysbiosis and subsequent GI dysfunction in the pathogenesis of ALS [22,23,24,25]. In parallel, human studies consistently report that the abundance and diversity of the intestinal flora in ALS patients are significantly altered when compared to healthy or other neurodegenerative controls.

Alterations to the gut flora are typically examined via high-throughput sequencing techniques such as 16S rRNA sequencing and shotgun metagenomics. Bioinformatic analyses may also be used to assess the taxonomic composition or diversity of detected microbial communities in various samples, as well as across different patient groups. To date, the largest human gut profiling study was conducted by Guo et al. [26] and included a total of 185 participants. The fecal microbiome of ALS patients and unrelated healthy controls were longitudinally compared at three different time points. Two bacterial phyla, Firmicutes and Cyanobacteria, were significantly different in relative abundance between patients and controls at the first collection point (baseline) [26]. Adjustment for confounding factors (sex, age, and body mass index) further highlighted alterations to the abundance of six specific genera: Bacteroides, Parasutterella, and Lactococcus were all significantly enriched in ALS samples, while Faecalibacterium and Bifidobacterium were markedly reduced compared to controls. The presence of a distinct gut microbiome in ALS patients was further validated by significant differences in the beta-diversity between the two study groups. Interestingly, only the abundance of Firmicutes significantly varied at the second time point [26], suggesting that the gut microbiome may continue to undergo changes over the course of the disease. In tandem, the relative abundance of Bacteroidetes and Firmicutes was significantly different at the first and second time points, respectively, in ALS patients with bulbar versus limb onset. Separate cohorts, albeit with some overlap in study participants, were also used to assess if any associations between dysbiosis and plasma metabolites were present [26,27]. Indeed, several microbes, such as Akkermansia muciniphila and members of the Lachnospiraceae family were correlated with alterations in plasma lipid-related metabolites [26]. Mendelian randomization analysis described acylcarnitine, bile acid, and fatty acid metabolism as potentially causal to ALS, and several acylcarnitines were further negatively correlated with scores on the revised ALS functional rating scale (ALSFRS-R). Taken all together, the microbiome-metabolome interface offers a promising framework for understanding the disease mechanisms underlying ALS and exploring new therapeutic interventions.

Other human microbiome studies also provide evidence of dysbiosis in ALS patients. For instance, Fang et al. [28] demonstrated several significant alterations to the gut microbiome at different taxonomic levels. Bacteroidetes (phylum), Bacteroidia (class), Bacteroidales (order), and Dorea (genus) were all enriched in ALS samples compared to healthy controls, whereas Firmicutes (phylum), Clostridia (class), Lachnospiraceae (family), Oscillibacter (genus), and Anaerostipes (genus) were notably decreased. Di Gioia et al. [29] reported that Escherichia coli, Clostridiales Family XI (family), Gastranaerophilalaes (family), and Cyanobacteria (phylum) were all significantly elevated in ALS, while Clostridiaceae 1 (family) was lower in patients than controls. While the total bacterial count did not differ between the two study groups, ALS stool samples showed lower DNA concentrations compared to controls, possibly due to significantly decreased amounts of yeast [29]. Patients with reduced yeast counts were significantly correlated with lower ALSFRS-R scores and forced vital capacity percentage (FVC%). Thus, shifts in various microbial populations, not only bacteria, may impact disease progression or clinical manifestation. Consistent with these findings, Zhai et al. [30] showed ALS stool samples had a significant increase in the relative abundance of Euryarchaeota (phylum), Methanobacteria (class), and Methanobrevibacter (genus) while Faecalibacterium (genus) and Bacteroides (genus) were reduced. ALS patients were also noted to have less rich and even microbial communities compared to healthy controls [30], which could impact metabolic function. Indeed, spectrophotometry showed that elevated fecal levels of SCFAs, nitrogen-containing compounds (NO₂-N/NO₃-N), and γ-aminobutyric acid were observed in ALS samples compared to controls. While not statistically significant, alterations in fecal metabolites may be a sign of underlying ALS-related GI dysfunction [30].

Given that caregivers who live or closely interact with ALS patients may share environmental exposures, and changes to the gut microbiome by extension, several studies use healthy family members and spouses as controls. Niccolai et al. [31], for example, compared the stool samples of ALS patients with those collected from cohabiting controls and found they were distinct. Patient samples showed a significant enhancement in the relative abundance of Senegalimassilia (genus), while Subdoligranulum (genus) and several members of Lachnospiraceae (family) were instead only elevated in family members. Among others, Adlercreutzia, Lachnospiraceae_FCS020_group, and Romboutsia were further correlated with disease progression and survival in the ALS group [31]. Patients with a slow progression rate, in particular, were associated with a higher abundance of Streptococcaceae (family) but a significant reduction in fecal α-diversity [31]. In contrast, Ngo et al. [32] reported no significant differences were found in the fecal microbiota of ALS subjects compared to the control group, which included healthy spouses, friends, and family members. Moreover, no associations between the gut microbiome and disease severity or site of onset were found. Anthropometric, metabolic, and clinical features of the disease were all described as being independent of the gut’s microbial composition [32]. Consistent with the previous study, however, the authors observed that an increase in the α-diversity of fecal microbiomes was associated with accelerated disease progression and a greater risk of early death [32]. This finding suggests that the contribution of gut dysbiosis to ALS pathogenesis may extend beyond changes to microbial composition to involve alterations in community diversity as well.

While most human microbiome studies present evidence of gut dysbiosis in ALS patients, some conflicting findings have been reported. Inconsistencies with regards to differences in the alpha/beta-diversity or Firmicutes/Bacteroidetes (F/B) ratio are amongst the most common. The F/B ratio is widely accepted as a sign of intestinal homeostasis and overall gut health, and its alteration has been previously described in inflammatory bowel diseases [33,34] as well as metabolic disorders [35,36,37]. However, the precise implications or effects of F/B imbalance on disease progression and patient outcome in ALS remain unknown due to conflicting results amongst current profiling studies. Some studies have reported that ALS patients exhibit significant shifts in the gut microbiome in favor of either Firmicutes [30,32,38] or Bacteroides [28,31,39,40], while others failed to detect any difference in the F/B ratio between ALS patients and controls [41]. These inconsistencies likely arise due to several reasons: heterogeneity in the subtypes, segment onset, and disease stage of ALS can all lead to varying differences in the gut microbial composition among study participants, while patient demographics, limited sample sizes, and methodological differences may also influence results. Addressing these limitations in future studies is pivotal to understanding the complex role of gut dysbiosis in ALS pathogenesis.

All currently published oral and fecal microbiome studies performed on human ALS patients are summarized in Table 1. It is important to note that evidence of dysbiosis and microbial translocation has also been found in other primary samples and/or tissues [42,43,44]. For example, in a recently published study by Liu et al. [42], nasal swabs collected from 66 ALS patients and 40 healthy caregivers differed significantly in microbial composition and diversity. At the phylum level, Bacteroidetes and Firmicutes were substantially increased in healthy nasal communities, while Actinobacteria dominated the nasal microbiota of ALS patients. Specific genera like Gaiella, Sphingomonas, Lachnospiraceae, and Klebsiella were all found to be significant predictors of ALS [42]. Faecalibacterium and Alistipes were also notably enriched in ALS patients and positively correlated with ALSFRS-R scores [42]. Further profiling and correlation analyses linked several changes to the nasal microbiota with ALS-related immune dysregulation and metabolic dysfunction. However, it is still unknown whether these associations are causal and if they significantly contribute to disease development. Another study by Ellis et al. [43] sought to characterize the microbial profile and total DNA content in the peripheral blood of ALS patients. Compared to healthy controls and multiple sclerosis (MS) patients, samples drawn from ALS subjects exhibited a significant reduction in Pseudomonas, Acidovorax, and Acinetobacter levels, with complete depletions of Funneliformis and Cloacibacterium [43]. Hydrurus, a freshwater alga belonging to the phylum Ochrophyta, was unexpectedly enriched in all ALS samples but only a quarter of the control and MS sample pools [43]. Moreover, principal component analysis revealed the combination of β-proteobacteria, γ-proteobacteria, and Ochrophyta could effectively sort and distinguish ALS patients from other sample populations.

Changes to the gut microbiota can influence distant organs by direct (e.g., translocation) and indirect (e.g., immune dysregulation) means. While the precise mechanisms are still under investigation, several connections between gut dysbiosis and ALS pathology have been identified (see Figure 1). It is important to highlight that further research is needed to definitively determine whether these findings are causal.

Given that GBA dysfunction and dysbiosis play a significant role in the pathogenesis of ALS, interventions and treatment strategies that target the gut microbiota may be of therapeutic benefit. Gut-modulating agents are broadly categorized into four main types: prebiotics, probiotics, postbiotics, and synbiotics. The use of these compounds in neurodegenerative disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), have previously proven effective in reversing dysbiosis and alleviating disease symptoms [78,79,80,81]. The following sections summarize all currently available preclinical evidence on the use of gut-microbiota-modulating agents in ALS.

Taken all together, recent evidence suggests a role for gastrointestinal dysfunction and gut dysbiosis in the pathogenesis of neurodegenerative disorders. Human microbiome studies consistently report that ALS patients exhibit distinct changes to their gut microbial composition and diversity. Drastic shifts in the microbial profile not only exacerbate local intestinal inflammation but can also promote chronic neuroinflammation, a hallmark of ALS pathology. Moreover, compromised gut barrier integrity, metabolic dysfunction, and immune dysregulation following dysbiosis have been linked to GBA dysfunction in ALS. Given these connections, the therapeutic potential of gut-modulating agents (prebiotics, probiotics, and postbiotics) have become a focus of recent research efforts. These interventions showed promising gut modulatory effects and neuroprotective properties in animal models of ALS. Depending on when they were administered, several agents were also able to rescue motor symptoms, or even mitigate disease progression. Although many parameters of gut health were assessed (e.g., fecal metabolites, inflammatory markers, and intestinal permeability), a major limitation of the preclinical studies summarized in this review is that the composition of gut microbiota following treatment was not always reported. It is, therefore, difficult to determine whether the effects of gut-modulating agents are secondary to the restoration of a healthy microbial balance without further research. Future studies should also continue exploring ALS-related gut dysbiosis to establish which mechanisms are potentially causal to disease pathology. To limit the possibility of any confounding factors from independently affecting both ALS and the gut microbiome, rigorous study designs are necessary. Lastly, translating these findings to human patients remains a challenge. Preclinical studies often use transgenic mice with SOD1 mutations, and while instrumental in our understanding of ALS pathology, these models do not fully represent the ALS patient population. It is important to investigate whether neuroprotection following gut modulation is reproducible in a variety of genetic and sporadic ALS models. Well-designed, large-scale randomized clinical trials are needed to better determine the efficacy and safety of these agents in ALS patients. Future studies should also aim to explore how individual variations in the human gut microbiome impact disease progression and treatment outcomes. Understanding the influence of genetic, dietary, and environmental factors could pave the way for more personalized treatment strategies.