When Kevin Eggan of Harvard University decided to raise some C9ORF72-deficient mice down the road at MIT’s Broad Institute, he had little inkling of the can of worms—or microbes—that he was about to open. At Harvard, the C9-deficient mice spent short lives ravaged by a systemic autoimmunity but, lo and behold, in their environs at the Broad, the same strain of mice bustled about as long as controls. On May 13 in Nature, Eggan and colleagues tied this contrast to distinct gut microbiomes colonizing the animals in the two respective facilities. The Harvard-raised mice got better with antibiotics, or with fecal microbes from Broad-reared mice. The study helps explain why C9ORF72 knockouts engineered at different labs around the country have exhibited strangely inconsistent phenotypes, and raises questions about whether the gut microbiome has something to do with the incomplete penetrance of the C9ORF72 expansion in people.
- In Harvard labs, C9 knockouts succumbed to an autoimmune disorder.
- At the Broad Institute, they lived normal lifespans despite some inflammation.
- Different gut microbiomes explained this variance.
The study implicates the microbiome as a potentially important, and previously underappreciated, modulator of ALS, wrote Eran Elinav of the Weizmann Institute of Science in Israel. “Understanding the molecular basis of host-microbiome interactions and their effects on ALS could lead to identification of new therapeutic targets of this deadly disease.”
“The variability of phenotype [of C9ORF72-deficient mice] has confused the field and has remained unresolved,” Aaron Gitler of Stanford University wrote to Alzforum. “In the current manuscript, the Eggan team seems to have solved this mystery and, in one fell swoop, also suggest a novel therapeutic strategy.”
Since tying hexanucleotide expansions in the first intron of the C9ORF72 gene to ALS/FTD in 2010, researchers have tried to work out the mechanisms involved (Sep 2010 news; Sep 2011 news). Three appear implicated: loss of function caused by hobbled expression of normal C9ORF72, gain of function related to extended repeat mRNAs, and polydipeptide repeats that arise from aberrant translation of the same. To focus on the loss of function, Eggan and several other groups generated knockouts. Across models and labs, these C9ORF72-deficient strains showed similar signs of age-related inflammation, such as swollen spleens, elevated pro-inflammatory cytokines, and an abundance of circulating immune cells. Beyond that, though, striking differences emerged. In Eggan’s mice, loss of one or both C9ORF72 alleles meant an early death, whereas another group saw that only in mice missing both alleles and another reported normal lifespan for both homo- and heterozygous knockouts (Jul 2016 news; Oct 2015 conference news; Mar 2016 news). Some researchers chalked up this variation to environmental differences, but what were they?
The Gut Factor. Variations in gut microflora influence inflammatory phenotypes in C9ORF72-deficient mice living in different facilities. [Courtesy of Fang and Hsiao, Nature News and Views, 2020.]
As it turned out, first author Aaron Burberry and colleagues decided to establish a C9ORF72-deficient mouse colony at the Broad Institute to take advantage of that facility’s set-up for behavioral studies. In a shocker, the mice at the Broad appeared normal from birth. Furthermore, they lived as long as control mice. The researchers established age-matched cohorts of mice at Harvard and the Broad to track their phenotypes. While the Harvard mice had sky-high levels of numerous pro-inflammatory cytokines in their blood, antibodies to self antigens, more neutrophils, fewer platelets, and enlarged spleens, all these phenotypes were much milder in the Broad mice, although the homozygous knockouts still had modestly enlarged spleens. By 37 weeks of age, the Harvard mice promptly fell off a rotating beam—a motor deficit that Eggan attributes to raging inflammation, as opposed to motor neuron degeneration. Broad mice stayed on the beam as long as control mice.
What about the Broad’s environment dampened these responses? Conditions at the two facilities—including diet and light cycle—were nearly identical. However, microbial screening reports were not. Compared with mouse poop collected at the Broad, droppings from mice at Harvard contained more immune-stimulating microbes, including murine norovirus, Helicobacter species, Pasteurella pneumotropica, and Tritrichomonas muris. Though elevated, levels of these microbes were still within a normal, non-pathogenic range.
If bacteria were to blame for the ill fate of the Harvard C9-deficient mice, might antibiotics save them? Indeed, treatment of the Harvard C9 knockouts with broad-spectrum antibiotics throughout their lives prevented the emergence of all inflammatory phenotypes. Antibiotic treatment even reversed symptoms of inflammation, including the enlarged spleens, when given to older C9ORF72-deficient mice that were already sick, and improved their balance on the rotating beam. “I was blown away by this,” said Eggan, a self-described critic of rodent microbiome research.
Bestowing Harvard mice with poop from Broad mice via oral gavage also lessened all their autoimmune woes. Microbial profiling confirmed that their gut microbiomes had been transformed into a Broad mouse-like profile.
Lest this turn into petty Harvard-Broad rivalry, could microbial profiles explain the variability in C9-deficient phenotypes reported by other labs? To find out, the researchers put out a call for mouse droppings from other facilities. They profiled microbes from Johns Hopkins University, Baltimore, where C9 knockouts died young, and from the Jackson Labs, Bar Harbor, Maine, where they lived normal lifespans. Strikingly, they found similarities between microflora from the “pro-survival” Broad and Jackson facilities, while the composition of gut microbes from the two “pro-inflammatory” Harvard and Johns Hopkins environments clustered together, as well. The findings strengthened support for the idea that microbes in the gut toggle the severity of inflammation in C9-deficient mice. The researchers are continuing to sample gut microbiota from other facilities housing C9 mice.
Does this to-do about mouse poop have anything to do with ALS? The researchers assessed how gut microbiota influenced inflammation in the spinal cords of the mice, where infiltrating immune cells wreak havoc in people with this disease. In C9 knockouts raised at Harvard, the researchers found abundant myeloid cells infiltrating the spinal cord. Lifelong treatment with antibiotics prevented the invasion.
There were microglial effects also. In mice at Harvard, microglia in the spinal cord expressed high levels of lysosomal markers, in agreement with previous reports tying C9 deficiency to problems with endolysosomal trafficking (Ugolino et al., 2016; Feb 2018 news). Microglia also expressed activation markers. Antibiotic treatment assuaged microglial activation, but did not reduce lysosomal markers. The findings suggested that gut microbiota hold sway over some, but not all, consequences of C9 loss in microglia.
Eggan said the phenotypes in the various mice are a product of the net effect of C9 loss of function, which makes myeloid cells more sensitive to inflammatory stimuli, and the presence of certain immune-stimulating microbes, which kicks these cantankerous cells into attack mode.
Robert Baloh of Cedars-Sinai Medical Center in Los Angeles said the study provides clear evidence that the gut microbiome influences how severe inflammation triggered by C9 deficiency will get. In Baloh’s lab, similarly generated C9-deficient mice developed telltale signs of systemic inflammation, including an enlarged spleen and lymph nodes, but lived a normal lifespan (O’Rourke et al., 2016). Baloh views the various phenotypes reported by different labs as part of an inflammatory spectrum, ranging from systemic inflammation to autoimmune attack. “It’s really about the severity of the inflammatory response, rather than a bunch of totally different phenotypes,” he said.
Baloh noted that immune-related phenotypes are notoriously sensitive to environmental differences, including gut microflora. Florent Ginhoux of the Agency for Science, Technology and Research in Singapore agreed, noting that the cancer immunotherapy field, in particular, has been dogged by variable findings among facilities. He said that the microbiome should be taken into account when comparing study results.
C9-deficient mice do not exhibit motor symptoms of ALS, nor do they harbor RNA foci or dipeptide repeat inclusions found in people with hexanucleotide expansions in the gene. Still, Eggan said that under certain conditions, loss of C9ORF72 function can promote a powerful neuroinflammatory response, which is a key component of human ALS. He also noted that in the SOD1 mouse model of ALS, treatment with antibiotics exacerbated disease, suggesting that each pathogenic mutation has a unique relationship with gut microbiota (Blacher et al., 2019). For C9 carriers, differences in gut microbiota could help explain the wide variation in disease onset, or even incomplete penetrance, he said.
Gitler agreed. “These results are tremendously exciting and surprising, and might help to explain the variable phenotype of C9ORF72 mutations in humans,” he wrote. “They could suggest that antibiotics could facilitate therapeutic efforts to treating (or preventing) disease caused by C9ORF72 mutations.”
Stanley Appel of Houston Methodist Neurological Institute in Texas said the paper beautifully documents how gut microbiota influence systemic inflammatory responses that exacerbate neurodegenerative disease. However, he questioned how the findings relate to ALS, given the lack of motor neuron degeneration in the C9-deficient mice. He emphasized that ALS starts with insults in motor neurons, which in turn instigate damaging neuroinflammation. Therefore, he said that while gut microbiota may aggravate disease, they are unlikely to dictate onset or penetrance among C9 carriers.
Eggan and other researchers are investigating these questions with a large, NIH-funded, natural history study of C9ORF72 carriers. They will ask whether gut microbiomes differentiate between carriers who develop the disease early, late, or not at all.
“These are obviously highly intriguing findings with profound implications for the treatment of ALS and FTLD based on modifying the gut microbiome,” wrote Nanda Kumar, Deepak Kumar, and Rudolph Tanzi of Massachusetts General Hospital in Boston. They noted that the findings could have relevance for other neurodegenerative diseases including AD and PD, where gut microbiota have also been implicated (Dec 2016 news; Feb 2017 news; Apr 2020 conference news). They believe future studies should consider how antibiotic treatments impact not only bacteria, but also fungi and other microbial inhabitants in the gut.
In their editorial accompanying the paper, Ping Fang and Elaine Y. Hsiao of the University of California, Los Angeles, wrote that more research is needed to zero in on the particular microbes and microbial functions that sway inflammation outside of the CNS, and to assess whether this peripheral inflammation influences the degeneration of motor neurons. “Unravelling [these mechanisms] would advance our understanding of the interactions between environmental factors and genetic risk factors in ALS, and might lead to new targets for clinical intervention.”—Jessica Shugart
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