SAMANTHA PRYOR, ND
Since this is my first article submission to NDNR, I would like to take a moment to tell you a little about myself. As I write this, I am currently employed as the last resident of the University of Bridgeport School of Naturopathic Medicine (UBSNM), which is also my alma mater. It saddens me deeply that the class of 2022 will be UBSNM’s final class, that our program will be closed.
On an even more personal note, though, the topic of this article – amyotrophic lateral sclerosis (ALS) – is a disease that has touched me to my core. I lost my father to this disease, at the young age of 51, between my first and second year of naturopathic medical school. He was diagnosed during my first year of school. As you might imagine, that started a process of continually learning more and more about this disease. Having spent so many hours researching ALS, I feel that I am becoming an expert. So, with that introduction, let us begin an exploration into microbiome alterations in ALS.
Gut-Brain Connection in ALS
Amyotrophic lateral sclerosis, also called motor neuron disease in other countries, is a neuromuscular disease that begins in the central nervous system with inflammation that damages the upper motor neurons. This damage leads to muscular weakness and eventual paralysis of the voluntary muscles in the body. The average life expectancy after diagnosis is 2 to 5 years. However, ALS can progress even faster than this, as was the case with my father who was diagnosed in March 2016 and passed away in June 2016. He died with respiratory failure, which is the ultimate cause of death in all ALS patients without adequate intervention.
It comes as no surprise to naturopathic doctors that the neuroinflammation in ALS can begin in the gut, similar to many other disease processes. My wise professor, Dr Eugene Zampieron, introduced this concept to our class in my first year of school: “If you are not sure where to begin treatment, start with the gut.” In addition, the microbiome is the new frontier in medicine, and research is uncovering new information every day. As recently as October 23, 2020, the AANP’s News Digest included an article entitled “Harvard Scientists Identify Gut-Brain Connection in ALS.” So, with that introduction, let us explore the research behind this article.
C9orf72 Mutation & ALS
The most prevalent cause of familial ALS is a genetic mutation called C9orf72; it can also contribute to familial frontotemporal dementia (FTD). This mutation, a hexanucleotide repeat expansion, results in loss of function in the gene. Animal studies have demonstrated that reduced function in C9orf72 results in cytokine storm, neutrophilia, and neuroinflammation.1
The goal of this most recent study, by Burberry et al, was to determine why long-term survival of C9orf72-mutant mice varied so much in previous studies. The researchers believed that the environment was a contributing factor.1 To conduct the study, the scientists placed C9orf72-mutant mice into a new facility at the Broad Institute – referred to C9orf72(Broad) mice – while they continued to house a colony of C9orf72-mutant mice at the Harvard Biological Research infrastructure – referred to C9orf72(Harvard) mice.
In this study, the C9orf72(Harvard) mice were found to die much earlier than the C9orf72(Broad) mice (p=0.0179), even though the diet, light cycle, and many other features of the environment were similar in both locations. However, a microbial screening of the mice at the 2 facilities found that murine norovirus, Helicobacter spp, Pasteurella pneumotropica, and Tritrichomonas muris were significantly more common in the C9orf72(Harvard) mice than in the C9orf72(Broad) mice. This microflora is not considered pathogenic and is found to be consistent with normal health in control mice. However, previous research had shown Helicobacter spp to have immune-stimulating properties.2 The authors suspected that these characteristics in the gut flora might be the cause of the early mortality and inflammatory phenotype found in the C9orf72(Harvard) mice.1
The researchers continued the study by treating the C9orf72(Harvard) mice with antibiotics, which decreased the overall diversity of the gut flora, but also the concentration of Helicobacter spp. This resulted in suppression of the inflammatory phenotype found in C9orf72(Harvard) mice, and led the researchers to conclude that “signals derived from the gut bacteria promote inflammation and autoimmunity when C9orf72 function is diminished.”1
The experiment continued with the researchers evaluating the role of the antibiotics in reducing morbidity in the C9orf72(Harvard) mice. In a cohort of the C9orf72(Harvard) mice, they used transient antibiotics to remove the gut flora. They then administered fecal transplants from the proinflammatory C9orf72(Harvard) mice to half of these mice, and administered fecal transplants from pro-survival C9orf72(Broad) mice to the other half. The mice receiving fecal transplants from the pro-survival mice showed significant improvement in their inflammatory and autoimmune phenotypes, demonstrating that the gut flora influences clinical outcome.1
To investigate the influence of specific microorganisms on phenotype, the researchers then examined the microbiomes of 2 groups of proinflammatory mice and 2 groups of pro-survival mice. The proinflammatory mice were found to have significantly altered diversity of flora as well as measurable counts of Helicobacter spp. In the pro-survival mice, however, Helicobacter spp were not present. The researchers found similar microbiota in the proinflammatory and pro-survival mice, and they ruled out a protective effect of Akkermansia municiphila and any potential inflammatory effect of Tritrichomonas muris. They concluded that the microbiome alters the onset and progression of autoimmunity and neuroinflammation in C9orf72 mutation carriers even before the diagnosis of ALS or FTD.1
SOD1G93A Mutation & ALS
In the study described above, the authors looked for a protective effect of Akkermansia municiphila on the C9orf72 mice because of the study we will look at next.
A mutation in the gene for copper/zinc superoxide dismutase-1 (SOD1) was the first discovered cause of ALS. Currently, it is believed to contribute to about 10% of familial ALS cases, including some sporadic cases, so this mutation is important to study. Several different mutations can occur within the SOD1 gene. This particular study examined SOD1G93A, one of the more common variants that has been shown to have a toxic gain-of-function in the enzyme activity and result in injury to motor neurons.2
In a study by Blacher et al, the microbiome of SOD1G93A mice was depleted using broad-spectrum antibiotics at 40 days of age; another group of SOD1G93A mice was administered only water.3 Motor testing, which included the rotarod locomotor test, the hanging-wire grip test, and neurological scoring, showed poorer outcomes in the antibiotic-treated SOD1G93A mice as compared to the water-treated SOD1G93A mice. Furthermore, in contrast to the water-treated mice, the antibiotic-treated mice exhibited a significant exacerbation of motor abnormalities in the antibiotic-treated mice throughout the entire disease course of ALS.4
The researchers also examined 2 groups of wild-type mice, one of which was administered broad-spectrum antibiotics. Performance in the rotarod locomotor test or the hanging-wire grip test was no different in the antibiotic-treated wild-type mice when compared to wild-type mice not given antibiotics, demonstrating that the antibiotics only exacerbated motor abnormalities in mice with SOD1G93A mutations. The investigators also found that growing wild-type and SOD1G93A mice in a germ-free setting resulted in higher mortality rates in the SOD1G93A mice as compared to the wild type, indicating that microbial drivers affect the progression of ALS.4
In a subsequent phase of the study, the investigators used 16S rDNA sequencing to examine the fecal microbiome of both SOD1G93A mice and wild-type mice. Results demonstrated a significant difference between the microbiomes of these 2 groups, starting early and throughout the course of ALS. There was also a significant difference between the microbiomes of the SOD1G93A and the wild-type mice that were treated with antibiotics. When the fecal metagenomes were analyzed, the microbial genes encoding enzymes involved in tryptophan metabolism and the enzymes involved in nicotinamide (NAM) and nicotinate metabolism were significantly reduced in SOD1G93A mice as compared to the wild-type mice, despite no differences in the food, water, oxygen consumption, locomotion, or heat production between the groups of mice.4
In the SOD1G93A mice, the researchers tested 11 strains of microbes thought to contribute to the severity of ALS progression. SOD1G93A and wild-type mice that were pretreated with antibiotics were inoculated with oral treatments of each of the 11 strains, 6 days apart, for 15 treatments. Most of these microbial strains did not affect the ALS symptoms, except for Ruminococcus torques and Parabacteroides distasonis, which appeared to exacerbate the disease progression.4
Another microbe found to be altered only in the SOD1G93A mice was Akkermansia municiphila (AM). The population of AM in the SOD1G93A mice showed a gradual reduction along the course of the disease; in contrast, the population of AM in the wild-type mice stayed relatively constant. When the SOD1G93A mice were pretreated with antibiotics and then treated with a mono-cultured AM strain, performance in all the motor tests and neurological scoring improved. Treatment of SOD1G93A mice with AM also resulted in a significantly prolonged life span, as compared to SOD1G93A treated with other commensal species (to serve as a control). At 140 days of age, the AM-treated SOD1G93A mice showed less brain atrophy on magnetic resonance imaging (MRI).4
Investigating a possible role of microbiome-regulated metabolites in ALS, the researchers found 2 metabolites that were synthesized by the wild-type mice but not by the SOD1G93A mice: phenol sulfate and NAM. Administering phenol sulfate to SOD1G93A mice via a subcutaneously implanted slow-release pump had no effect on their ALS symptoms. The study found that anaerobically grown AM produced significantly higher levels of NAM than other commensal isolates. Metagenome analysis revealed that 8 out of 10 genes related to the AM genome and that encode enzymes participating in NAM metabolism were significantly enriched in AM-treated SOD1G93A mice when compared to vehicle-treated SOD1G93A mice.4
Administering NAM via a subcutaneously implanted slow-release pump led to improved motor test and neurological scoring in the NAM-treated SOD1G93A mice as compared to the vehicle-treated SOD1G93A mice. The researchers processed the spinal cord samples of AM- and NAM- treated mice for RNA-sequencing, and found that 31 genes were significantly changed in both the AM-treated mice and the NAM-treated SOD1G93A mice. Some areas were particularly enriched by treatment with AM and NAM: mitochondrial structure and function, nicotinamide adenine dinucleotide+ (NAD+) homeostasis, and removal of superoxide radicals – all areas known to be impacted in ALS. There was a significant change found in 28.6% of the promoters of genes that share a binding site for the transcription factor nuclear respiratory factor 1 (NRF-1), which is involved in the biogenesis of mitochondria and its function in respiration.4
Lastly, the researchers collected stool samples from 37 ALS patients and 29 healthy family members (serving as controls) and examined them using metagenomic sequencing. The ALS patients’ microbiomes showed significant differences in bacterial gene content, including a decrease in several genes involved in the metabolism of tryptophan and NAM when compared to healthy controls; many of these genes mapped to the AM genome.4 These human stool findings from patients with ALS support the findings in the SOD1G93A mice, suggesting that AM – which increases NAM – is an important microbe in ALS patients.
Conclusion
As evidenced by the research discussed here, the microbiome is important in all patients with ALS. How I wish this research had been available when my father was diagnosed with the disease. For your ALS patients, I recommend running a stool analysis that uses PCR in order to evaluate bacterial diversity. Correcting the diversity helps ensure a balance among all 6 primary phyla tested. High amounts of Proteobacteria must be lowered, since this phylum contains the Helicobacter genus, which was shown in the first study to aggravate mice with the C9orf72 mutation. Work to increase all of the short-chain fatty acid producers if any are found to be deficient. Treat any pathogenic infections found in the analysis. Lastly, look at the Verrucomicrobia phylum and increase the Akkermansia muciniphila if this is low, due to its role in nicotinamide production and overall improvement in gut health.
References
- Burberry A, Wells M, Limone F, et al. C9orf72 suppresses systemic and neural inflammation induced by gut bacteria. Nature. 2020;582(6):89-94.
- Whary MT, Fox JG. Natural and experimental Helicobacter infections. Comp Med. 2004;54(2):128-158.
- Borchelt DR, Lee MK, Slunt HS, et al. Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral scelerosis posesses significant activity. Proc Natl Acad Sci U S A. 1994;91(17):8292-8296.
- Blacher E, Bashiardes S, Shapiro H, et al. Potential roles of gut microbiome and metabolites in modulating ALS in mice. Nature. 2019;572(8):474-480.
Samantha Pryor, ND graduated from UBCNM in 2019. Currently, she is completing a 1.5-year residency at her alma mater, which will finish in spring of 2021. Serving 6 years in the United States Army as a medic prepared her for the rigors of naturopathic school, where she excelled and graduated at the top of her class. Specializing in Generative Medicine and nature cure, Dr. Pryor is excited to see where life takes her next.
