. C9ORF72 suppresses systemic and neural inflammation induced by gut bacteria. Nature. May 13, 2020.

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  1. This new manuscript from Kevin Eggan and colleagues is surprising and remarkably important, with direct implications for therapies that are currently in development for ALS and FTD. Mutations in the C9ORF72 gene are the most common cause of ALS. This discovery has completely revolutionized the ALS research field. Intense efforts are now focused on understanding the mechanism(s) by which C9ORF72 mutations cause disease and developing therapeutic strategies to treat patients harboring C9ORF72 mutations.

    The mutation is a GGGGCC hexanucleotide repeat expansion. This is thought to contribute to ALS through a combination of reduced C9ORF72 expression and the production of toxic RNAs or dipeptide repeat proteins from the repetitive RNA. Unlike other, more fully penetrant, ALS disease genes (e.g., SOD1), the penetrance of C9ORF72 mutations is variable. Some individuals with these mutations develop ALS, some develop frontotemporal dementia, and others are unaffected. Adding complexity, it has emerged that individuals with C9ORF72 mutations have increased susceptibility to autoimmune disorders, like lupus and multiple sclerosis.

    To explore the normal function of C9ORF72, and to define the potential role of reduced C9ORF72 function in disease, in the last few years researchers in the ALS field (including the Eggan team) have generated several independent lines of C9ORF72 +/- and -/- knockout mice. Perplexingly, these mice, some of which were generated using the same targeted alleles on the same genetic background, presented with variable phenotypes depending on the group. For example, the Baloh team’s knockout at Cedar’s Sinai in Los Angeles presented with splenomegaly, autoimmunity, and no shortened lifespan, whereas the Eggan knockout mouse at Harvard in Boston presented with the splenomegaly, autoimmunity, but with a shortened lifespan. This variability of phenotype has confused the field and has remained unresolved.

    In the current manuscript, the Eggan team seems to have solved this mystery and, in one fell swoop, also suggests a novel therapeutic strategy.

    The authors start by re-deriving their original knockout mouse, but this time in the Broad Institute, in a different mouse facility. They were surprised to find that the same line of C9ORF72 knockouts, now at the Broad, did not have the shortened lifespan like they did at Harvard. This launched a mission to figure out what it was in the Harvard facility (or not in the Broad facility) that was causing the difference in lifespan. Even though both facilities were employing high standards for animal care and cleanliness, the authors noted that the Harvard facility had a higher level of some bacteria and viruses than the Broad one. They hypothesized that perhaps the reduced expression of C9ORF72 in mouse could make them more sensitive to bacterial and viral infections, and subsequent pro-inflammatory phenotypes, which could explain the differential phenotypes.

    To test this hypothesis, they treated the Harvard housed +/- and -/- mice with broad spectrum antibiotics. Remarkably, this was sufficient to ameliorate phenotypes in the Harvard mice! Thus, the presence of a pro-inflammatory microbial burden contributes to reduced lifespan in the C9ORF72 KO mouse, while a C9ORF72 KO strain re-derived and bred in aseptic environment has a normal lifespan. 

    There are several extremely compelling experimental results in this paper. Among them are that the authors were able to obtain samples from colonies in other institutions that either had early mortality in the knockout mouse (C9Harvard, Johns Hopkins) or normal survival (C9Broad, Jackson labs). After additional microbial profiling of these new samples, the authors found differential expression of bacterial species along the lines of early mortality/pro-inflammatory cohorts and pro-survival cohorts. These results strengthen the authors’ claims that the gut microflora can influence the survival of C9 knockout mouse, as the findings are no longer restricted to the authors’ colonies. 

    The other key experiment showed that fecal transplantation from the pro-survival Broad colony to the knockout C9Harvard mice can dampen the pro-inflammatory immune reaction in the knockout mice, compared to fecal transplantation from the Harvard colony to the knockout C9Harvard mice. This again supports the authors’ central claim that the environment and gut microbial context can influence the pathological inflammatory response in the C9KO mice, and complements the broad-spectrum antibiotic experiments.

    Of course, the reverse experiment (fecal transplantation from Harvard to the C9Broad mice) and survival data would be desirable and perhaps an area for future consideration. The paper also leaves open the question about the potential for anti-inflammatory, pro-survival microbial species present in the Broad/Jackson cohorts.

    Collectively, these results are tremendously exciting and surprising. They might help to explain the variable phenotype of C9ORF72 mutations in humans, and perhaps suggest that antibiotics could facilitate therapeutic efforts to treating (or preventing) disease caused by C9ORF72 mutations.

    View all comments by Aaron Gitler
  2. Using a mutant C9ORF72 mouse model, Burberry et al. report on the impact of the gut microbiome on disease progression in ALS. They show that fecal transplant using gut microflora from mice with loss of both (-/-) alleles of C9ORF72 raised in a protective environment (Broad Institute, MIT) ameliorated disease pathology when introduced into mutant mice raised in an inflammation-prone environment (Harvard University). Apart from having a shorter lifespan, C9ORF72 mutant mice raised at the Harvard facility showed increased levels of pro-inflammatory signals and autoantibodies, higher neutrophil infiltration, and lower platelet counts, as compared to mice raised at the Broad Institute.

    Previous studies have reported profound effects of the gut microbiome on Alzheimer’s disease (AD) and Parkinson disease (PD) phenotypes (Minter et al., 2017Sampson et al., 2016; Harach et al., 2017). Here, the authors found different microbial compositions under different environmental conditions that profoundly influence the C9ORF72 mutant phenotype and survival rate, as well as CNS recruitment of peripheral myeloid progenitors.

    These are obviously highly intriguing findings with profound implications for the treatment of ALS and FTLD based on modifying the gut microbiome. However, with regard to the finding that broad-spectrum antibiotics significantly reduced inflammatory and autoimmune phenotypes in the mutant mice, it will be important in future studies to also compare the gut mycobiome profiles of these two groups of mice.

    The gut environment is a symbiotic ecosystem that requires cooperative interaction among all microbial groups. Prolonged antibiotic use in mice would likely promote fungal overgrowth, which can be harmful or beneficial depending on the competing species. While the mycobiome remains one of the most understudied areas of the gut microbiome, their synergistic coexistence in the gut ecosystem, and recent reports of their link to other neurodegenerative disease such as AD and PD, underscore their importance in promoting overall gut health. 

    Therefore, correlation of mycobiome modulation pre- and post- antibiotic administration, and post-FMT will be very interesting to investigate in future studies.

    References:

    . Interactions between the microbiota, immune and nervous systems in health and disease. Nat Neurosci. 2017 Feb;20(2):145-155. Epub 2017 Jan 16 PubMed.

    . Antibiotic-induced perturbations in microbial diversity during post-natal development alters amyloid pathology in an aged APPSWE /PS1ΔE9 murine model of Alzheimer's disease. Sci Rep. 2017 Sep 5;7(1):10411. PubMed.

    . Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson's Disease. Cell. 2016 Dec 1;167(6):1469-1480.e12. PubMed.

    . Reduction of Abeta amyloid pathology in APPPS1 transgenic mice in the absence of gut microbiota. Sci Rep. 2017 Feb 8;7:41802. PubMed.

    . Potential roles of gut microbiome and metabolites in modulating ALS in mice. Nature. 2019 Aug;572(7770):474-480. Epub 2019 Jul 22 PubMed.

    View all comments by Rudy Tanzi
  3. We have read with great interest the important new paper from Burberry and colleagues demonstrating the influence of gut bacteria on the ability of the C9ORF72 hexanucleotide repeat to suppress inflammation in the brain (Burberry et al., 2020). We agree that it will be of value to also evaluate the contribution of fungi, as suggested by  Shanmugam, Kumar and Tanzi.

    In addition, it is important to consider where the interaction between the microbiota and the host is taking place. The oral cavity is home to a vibrant microbiome, which has profound influences on the immune and nervous systems. It has been proposed that the site at which this interaction occurs influences the initial phenotype of the neurodegenerative disorder, e.g. oral microbes may lead to bulbar ALS, and colonic microbes may lead to spinal onset (Friedland and Chapman, 2017). In order to understand the variable phenotypes of the C9ORF72 related disorders we need to comprehensively consider the microbial agents involved, as well as where the microbial-host interaction is taking place.

    References:

    . C9ORF72 suppresses systemic and neural inflammation induced by gut bacteria. Nature. May 13, 2020.

    . The role of microbial amyloid in neurodegeneration. PLoS Pathog. 2017 Dec;13(12):e1006654. Epub 2017 Dec 21 PubMed.

    View all comments by Zimple Kurlawala

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