Harvard University scientists have identified a new gut-brain connection in the neurodegenerative disease amyotrophic lateral sclerosis, or ALS.
The researchers found that in mice with a common ALS genetic mutation, changing the gut microbiome using antibiotics or fecal transplants could prevent or improve disease symptoms.
Published in the journal Nature, the findings provide a potential explanation for why only some individuals carrying the mutation develop ALS. They also point to a possible therapeutic approach based on the microbiome.
“Our study focused on the most commonly mutated gene in patients with ALS. We made the remarkable discovery that the same mouse model — with identical genetics — had substantially different health outcomes at our different lab facilities,” said Kevin Eggan, Harvard professor of stem cell and regenerative biology.
“We traced the different outcomes to distinct gut microbial communities in these mice, and now have an intriguing hypothesis for why some individuals carrying this mutation develop ALS while others do not.”
Different facilities, different outcomes
The researchers initially studied the ALS genetic mutation by developing a mouse model at their Harvard lab facility. The mice had an overactive immune response, including inflammation in the nervous system and the rest of the body, which led to a shortened lifespan.
In order to run more detailed experiments, the researchers also developed the mouse model in their lab facility at the Broad Institute, where Eggan is the director of stem cell biology at the Stanley Center for Psychiatric Research. Unexpectedly, although the mice had the same genetic mutation, their health outcomes were dramatically different.
“Many of the inflammatory characteristics that we observed consistently and repeatedly in our Harvard facility mice weren’t present in the Broad facility mice. Even more strikingly, the Broad facility mice survived into old age,” said Aaron Burberry, postdoctoral fellow in the Eggan lab and lead author of the study.
“These observations sparked our endeavor to understand what about the two different environments could be contributing to these different outcomes.”
Searching the gut microbiome
Looking for environmental differences between the mice, the researchers honed in on the gut microbiome. By using DNA sequencing to identify gut bacteria, the researchers found specific microbes that were present in the Harvard facility mice but absent in the Broad facility mice, even though the lab conditions were standardized between facilities.
“At this point, we reached out to the broader scientific community, because many different groups have studied the same genetic mouse model and observed different outcomes,” Burberry said. “We collected microbiome samples from different labs and sequenced them. At institutions hundreds of miles apart, very similar gut microbes correlated with the extent of disease in these mice.”
The researchers then tested ways to change the microbiome and improve outcomes for the Harvard facility mice. By treating the Harvard facility mice with antibiotics or fecal transplants from the Broad facility mice, the researchers successfully decreased inflammation.
By investigating the connection between genetic and environmental factors in ALS, the researchers identified an important gut-brain connection. The gut microbiome could influence the severity of disease — whether individuals with the genetic mutation develop ALS, the releated condition frontotemporal dementia, or no symptoms at all — and could be a potential target for therapy.
“Our study provides new insights into the mechanisms underlying ALS, including how the most common ALS genetic mutation contributes to neural inflammation,” Eggan said. “The gut-brain axis has been implicated in a range of neurological conditions, including Parkinson’s disease and Alzheimer’s disease. Our results add weight to the importance of this connection.”
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects upper and lower motor neurons, resulting in muscle atrophy, respiratory failure and death (Brown and Al-Chalabi, 2017).
ALS risk factors include advanced age and certain genetic mutations; however, epigenetic mechanisms are also altered and may represent a link between genetic factors and life-long environmental exposures, which are also known to increase disease risk (Paez-Colasante et al., 2015; Su et al., 2016).
However, despite intense research, the full spectrum and temporal course of ALS risk factors remain unknown, as does the precise disease etiology. Although age, genetics and environmental factors play a role, disease onset likely results from the dynamic interconnectivity of a network of altered pathways over time.
Accumulating evidence suggests that the gut microbiome is important in ALS (McCombe et al., 2019). The intestinal flora in ALS mice expressing mutant human superoxide dismutase 1 (SOD1G93A) is distinct from wild-type (WT) animals, with greater intra-communal diversity, differences in specific microbial flora (Blacher et al., 2019) and fewer butyrate-producing bacteria (Wu et al., 2015).
Supplementing SOD1G93A animals with bacteria (Blacher et al., 2019) or a bacteria-derived metabolite, e.g. butyrate (Zhang et al., 2017), restores the animal’s gut microbiome and lengthens lifespan.
Furthermore, clinical studies report differences in fecal microbiota from ALS patients compared to healthy volunteers, suggesting a possible translation to humans (Mazzini et al., 2018; Rowin et al., 2017; Blacher et al., 2019).
Recent studies also implicate the immune system in ALS progression (Thonhoff et al., 2018). As in the microbiome, genetics and epigenome restructuring by environmental cues impact immunity (Anaya et al., 2016).
In ALS SOD1G93A mice, immune activation in the central nervous system (CNS) and peripheral nervous system is distinct compared to control animals, with an accumulation of multiple immune cell types associated with disease progression (Alexianu et al., 2001; Chiu et al., 2009).
Specific immune cell populations such as CD4T cells may be protective (Beers et al., 2008), whereas others, such as neutrophils, inflammatory monocytes, CD8T cells and innate lymphoid cells, are destructive (Finkelstein et al., 2011; Murdock et al., 2017; Butovsky et al., 2012; Coque et al., 2019).
In addition, the impact of the immune system may depend on the stage of disease, with protective cytokines, such as interleukin (IL)-4 and IL-10, expressed in the CNS during early disease, whereas pro-inflammatory cytokines, such as IL-6, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), are expressed during late disease (Henkel et al., 2006; Beers et al., 2011).
Finally, several studies also report immune changes in the peripheral blood of ALS patients compared to healthy controls. As in mouse models, CD4T cells appear to be protective, whereas other immune cells, such as neutrophils and monocytes, appear to accelerate disease (Murdock et al., 2017; Beers et al., 2018; Gustafson et al., 2017).
There are also emerging relationships among ALS risk factors. For example, the gut microbiome impacts host immunity (Rooks and Garrett, 2016) and vice versa (Kato et al., 2014), suggesting possible mutual regulation. Specifically, the intestines of SOD1G93A mice are populated by an increase in abnormal Paneth cells (Wu et al., 2015).
Paneth cells are specialized gut epithelial cells that are part of the host innate immune system and normally release antimicrobial peptides in response to bacterial pathogens. However, in SOD1G93A mice, they are defective and secrete lower levels of antimicrobial peptides, underscoring one mechanism through which the microbiome may influence the immune system in ALS.
In addition, as mentioned above, the gut flora of SOD1G93A mice produces less butyrate (Wu et al., 2015), which is a known inducer of differentiation for colonic Treg cells that help maintain tolerance to self-antigens and prevent autoimmune disorders (Furusawa et al., 2013), suggesting another path of convergence between microbiome and immune system.
The microbiome also exerts an effect on the epigenome via secretion of metabolites and immune modulation, even to sites remote from the gut such as the brain (El Aidy et al., 2016), leading to changes in the host epigenome, including DNA methylation, histone modifications and microRNA alterations (Qin and Wade, 2018).
In the context of ALS, the gut microbiome-epigenome connection has not been investigated. However, the epigenome is significantly perturbed in ALS, with differences in microRNA, global and loci-specific cytosine methylation (5mC) and hydroxymethylation (5hmC), and histone deacetylase mRNA levels in ALS patient spinal cord tissue compared to healthy individuals (Figueroa-Romero et al., 2016, 2012; Janssen et al., 2010).
Thus, in ALS, the gut microbiome, immune system and epigenome comprise a trio of dysregulated biological processes, the interconnection of which could shed light on disease progression.
In the current study, we systematically evaluated alterations in gut microbiome, immune system, and ileum and brain epigenetic (5mC, 5hmC) modifications in SOD1G93A mice at multiple time points relative to symptom onset, i.e. skeletal muscle loss and impaired locomotion.
We report new associations between muscle loss and neuromuscular weakness with the microbiome, immunophenotypes and epigenetic marks. Defining the evolution and interrelation of these processes may elucidate ALS pathomechanisms and guide our development of better biomarkers for earlier diagnosis and novel therapies for improved ALS patient survival.