Through the ages, the quest for longevity has taken alchemists and scientists to unusual places. A present-day foray led Meng Wang at Baylor College in Houston to explore the jungle of genes inside a bacterium that lives in the gut of a tiny worm. In the June 15 issue of Cell, Wang and colleagues report finding 29 Escherichia coli genes that, when deleted, lengthen the lives of their Caenorhabditis elegans hosts. The study also shows that 14 of these mutations help keep Aβ-expressing worms moving and living longer. Unraveling the mechanism underlying five of the mutations, the scientists found that the bacterial polysaccharide colanic acid lengthened the worm’s life by modulating mitochondrial signaling in their cells.
“It’s a brilliant paper, a milestone in the field. Combining the genetic versatility of the worm with the accessibility of E. coli genetics is very powerful,” said Dario Valenzano at the Max Planck Institute in Cologne, Germany, who recently extended the lifespan of middle-aged fish by re-colonizing their guts with bacteria from young fish (Smith et al., 2017).
Microbial gut-dwellers influence their hosts in many ways. They churn out essential metabolites, process food, and biochemically modify host metabolites. And a growing number of conditions, including aging and neurodegenerative disease, seem to be influenced by these bacterial communities. Experiments in C. elegans, a workhorse in the field of aging research, have pointed to bacterial variants and small molecules they secrete, such as non-coding RNAs and nitric oxide, as modulators of the worm’s lifespan (Heintz and Mair, 2014).
So far, no one had conducted a systematic, genome-wide search for bacterial genes that influenced C. elegans longevity. To do so, first author Bing Han screened a library of 3,983 E. coli mutants, each lacking a single, non-essential gene, for their effects on C. elegans. Twenty-nine mutants prolonged the worms’ lifespan by more than 10 percent. Top performer hns, which encodes a transcriptional regulator, made the worms live 40 percent longer. Many of the mutants were unable to prolong the lives of worms that already carried mutations in genes previously known to regulate lifespan, such as daf-16, rsks-1, raga-1, rict-1, or a mutation that mimics caloric restriction, suggesting they act through these pathways.
To see if these life-extending bacterial mutations affect age-related pathologies such as cancer and neurodegeneration, the researchers tested them in tumor-prone C. elegans mutants and in Aβ-expressing transgenic worms. The latter were developed by Chris Link at the University of Colorado in Boulder; they express Aβ42 under the control of the muscle-specific unc-54 promoter/enhancer (Link, 1995). Fourteen of the bacterial mutants prolonged the lives of these transgenic worms by 10 to 40 percent; 12 delayed their age-associated paralysis (see image). Interestingly, 13 of the 14 life-extending mutants also prolonged the lives of tumor-ridden worms.
Link praised the findings but cautioned that the paralysis in his worms confounds survival measurements. For example, some females die because their paralyzed vulval muscles render them unable to lay their eggs. He also noted that the transgenic protein, expressed only in muscle cells, forms mostly intracellular inclusions, rather than typical extracellular plaques.
Wang and colleagues then focused on two bacterial mutants, Δlon and Δhns, that extended lifespan in normal and Aβ-expressing worms, but did so independently of the known longevity pathways. They noticed that both lon and hns encode proteins that reduce levels of colanic acid (CA). Secreted by many gut bacteria, this polysaccharide contains a repeating unit of glucose, galactose, fucose, and glucuronic acid, decorated by pyruvate and acetate. Wang found that three other bacterial mutants also overproduce CA; they not only extend lifespan but dampen the mitochondrial fragmentation normally seen in aging body-wall muscles.
To test whether CA was indeed an anti-aging elixir, Wang’s team added purified CA to wild-type C. elegans. Though less potent than the mutations, a little dietary CA extended the lives of worms grown on a variety of bacteria. It made old worms move faster, and once again prolonged the lives of tumor-prone and Aβ-expressing worms. Even a distantly related species, Drosophila melanogaster, benefited from eating CA. The flies lived longer and were more active later in life than controls.
Probing how CA accomplishes this, the authors searched for pathways that, when mutated in C. elegans, would cancel out CA’s benefit. Indeed, two mutations that disrupt the mitochondrial electron transport chain negated CA’s effects. Wang and her colleagues also observed that mitochondria in the intestinal cells of worms eating CA were more fragmented than those of non-treated controls. This CA-induced splintering of mitochondria also happened in a mammalian fibroblast cell line.
Even though mitochondrial fragmentation in aging muscles of the worm’s body wall is considered a sign of age-associated atrophy, the researchers reasoned that the CA-induced fragmentation in gut cells and fibroblasts could reflect healthy cells ramping up a quality-control mechanism that rids cells of damaged or dysfunctional mitochondria. Drp-1 is a gene required for mitochondrial fission and linked with increased longevity. To test this, the authors added CA to drp-1 mutants, and as expected, CA was no longer able to promote longevity. Drilling deeper, the researchers found that in mutants with compromised mitochondrial protein folding, CA triggered a strong mitochondrial stress response, activating the unfolded protein response (UPRmt) via the ATFS-1 transcription factor.
“They dissected down to the mechanism in both the bug and the host,” said Valenzano. “They can recapitulate the effect with the metabolite and show that it’s robust in other species. It’s a super-strong story.”
Does CA help cells beyond the gut? “We think the major active site is the gut,” said Wang. The authors found that CA requires host endocytic genes rab-5 and rab-7 to lengthen lifespan, suggesting the intestinal cells need to endocytose CA for it to work. “The gut then generates second messengers that could reach distant tissues, like muscle.” Other studies show that inducing the UPRmt in one tissue can induce a mitochondrial stress response in a distant tissue (Durieux et al., 2011). “Mitochondria are known to be important mediators of long-distance signals,” said Wang.
What’s next for CA? Many unknowns remain. Culture bacteria produce CA in response to stress and biofilm formation, but how and when it is made in vivo is unknown, said Wang. The length of the polymers, and how much of it bacteria make and consume, are nagging questions, she added, because the bacterial mutants that overproduce CA are twice as effective at lengthening lifespan as the purified polymer. “We are fractionating CA based on molecular weight,” said Wang. This will help pinpoint the active ingredient(s) and enable dosing studies.
The scientists want to test CA in mammals. “We need to purify a lot to test in mice,” said Wang. “We are also working on attaching a particle to track it in vivo.” Asked if she has tried CA herself, Wang said, “We joke in the lab: Are you secretly taking it?” Turning serious, Wang said CA could become an appealing supplement because, given its source, she suspects it likely will be safe.
Does this work have implications for neurodegenerative disease? Taoufiq Harach of Reminisciences, a biotechnology company in Paris focusing on microbe-based treatment and diagnosis for Alzheimer’s disease, said he would have liked to see the effect of the life-extending mutants and CA on amyloid. He also noted the importance of examining other AD models that more closely mirror the human disease.
Link noted that the mitochondrial-CA connection fits with his unpublished observation that shifting the balance of mitochondrial fission versus fusion toward fission made Aβ-expressing worms more mobile. Knocking down drp-1 reduced mitochondrial fission, as expected, but also increased paralysis, whereas knocking down EAT-3, a gene required for mitochondrial fusion, got the worms moving. Link added that many mitochondria in the muscle cells are misshapen and depolarized, and he suspects amyloid may be poking holes in them. If so, increasing the organelle’s turnover may be beneficial. It is unclear how this scenario would unfold in the brain.
Researchers have drawn connections between gut bacteria and neuropathology. Microbiome changes have been reported early in neurodegeneration (e.g., Apr 2017 news; Dec 2016 news), and several studies have connected the microbiome to inflammation and neurodegeneration. One recent study linked brain amyloidosis to bacteria with known pro-inflammatory effects, while bacteria with known anti-inflammatory effects were reduced in elderly people with cognitive impairment (Cattaneo et al., 2017). There are also reports of bacterial short-chain fatty acids modulating inflammation.
Looking ahead at future clinical applications, some researchers are betting on probiotics while others are hunting down specific metabolites. Harach favors the former. He thinks isolating specific molecules and finding the right dosing will take more time and money than tweaking a person’s microbiome. He claimed to have identified bacterial strains that protect mice against amyloid; his company is looking for funding to begin a clinical trial. In a recent study, researchers laced the drinking water of 3xTg-AD mice with a cocktail of lactic acid bacteria and bifidobacteria. The treated mice had less amyloid aggregates and better performance on behavior assays than those drinking plain water. The authors suggest the effect may be mediated by partial restoration of proteasome and autophagy function (Bonfili et al., 2017).
On the other hand, Ullrich Wüllner at the University of Bonn in Germany is seeking specific bacterial molecules to help patients with Parkinson’s disease. He is interested in short-chain fatty acids and is conducting metabolomic analysis to find other therapeutic candidates, including anti-inflammatory compounds.
For her part, Wang’s team is trying to generate probiotic strains of the other longevity-promoting mutants for testing in mammals. She sees advantages in both approaches. Working with specific compound allows for targeted, mechanistically based interventions, but probiotics may have broad beneficial effects arising from the joint activities of multiple compounds that could be difficult to tease apart.—Marina Chicurel
- Rumblings of Parkinson’s: Gut Microbiome Shifts in Early Stage of Disease
- Do Microbes in the Gut Trigger Parkinson’s Disease?
Research Models Citations
- Smith P, Willemsen D, Popkes M, Metge F, Gandiwa E, Reichard M, Valenzano DR. Regulation of Life Span by the Gut Microbiota in The Short-Lived African Turquoise Killifish. BioRxiv, April 6, 2017
- Heintz C, Mair W. You are what you host: microbiome modulation of the aging process. Cell. 2014 Jan 30;156(3):408-11. PubMed.
- Link CD. Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9368-72. PubMed.
- Durieux J, Wolff S, Dillin A. The cell-non-autonomous nature of electron transport chain-mediated longevity. Cell. 2011 Jan 7;144(1):79-91. PubMed.
- Cattaneo A, Cattane N, Galluzzi S, Provasi S, Lopizzo N, Festari C, Ferrari C, Guerra UP, Paghera B, Muscio C, Bianchetti A, Volta GD, Turla M, Cotelli MS, Gennuso M, Prelle A, Zanetti O, Lussignoli G, Mirabile D, Bellandi D, Gentile S, Belotti G, Villani D, Harach T, Bolmont T, Padovani A, Boccardi M, Frisoni GB, INDIA-FBP Group. Association of brain amyloidosis with pro-inflammatory gut bacterial taxa and peripheral inflammation markers in cognitively impaired elderly. Neurobiol Aging. 2017 Jan;49:60-68. Epub 2016 Aug 31 PubMed.
- Bonfili L, Cecarini V, Berardi S, Scarpona S, Suchodolski JS, Nasuti C, Fiorini D, Boarelli MC, Rossi G, Eleuteri AM. Microbiota modulation counteracts Alzheimer's disease progression influencing neuronal proteolysis and gut hormones plasma levels. Sci Rep. 2017 May 25;7(1):2426. PubMed.
- Main BS, Minter MR. Microbial Immuno-Communication in Neurodegenerative Diseases. Front Neurosci. 2017;11:151. Epub 2017 Mar 23 PubMed.
- Han B, Sivaramakrishnan P, Lin CJ, Neve IA, He J, Tay LW, Sowa JN, Sizovs A, Du G, Wang J, Herman C, Wang MC. Microbial Genetic Composition Tunes Host Longevity. Cell. 2017 Jun 15;169(7):1249-1262.e13. PubMed.