09 May 2016

Microbes both friend and foe might contribute to the formation of amyloid plaques, according to researchers who spoke at the third annual Zilkha Symposium on Alzheimer Disease & Related Disorders, held April 15, 2016, in Los Angeles. Rudy Tanzi of Massachusetts General Hospital in Charlestown presented ongoing research on his and Robert Moir’s hypothesis that Aβ is an antimicrobial peptide, reporting that Salmonella bacteria can seed formation of plaques in the brains of mice. In turn, Sangram Sisodia of the University of Chicago focused on the bacteria that normally populate the gut. Shifting that teeming mass with intense antibiotic treatment reduced plaque burden in mice by half, he told attendees.

Making the case for a role for pathogens in AD, Tanzi noted that many of the genes recently found to be involved in Alzheimer’s disease risk function in the immune system. For example, high levels of TREM2 promote phagocytosis by microglia, while mutations in CD33 reduce phagocytosis rates, increasing amyloid plaque burden and overall Aβ load, Tanzi said.

Tanzi was the first to say his hypothesis warrants healthy skepticism. Referencing the Schopenhauer quote of the three stages of truth, whereby a novel idea is first ridiculed, then violently opposed, then finally accepted as self-evident, Tanzi placed his assertion that Aβ is an antimicrobial peptide between stages one and two. Meeting attendees were diplomatic, telling Alzforum the idea was intriguing and worthy of further investigation.

Anti-Microbial Aβ?
Antimicrobial peptides (AMPs) are charged peptides of 12-50 amino acids. “They are our first line of defense,” Tanzi said, explaining that these little peptides oligomerize into a “nanonet” of fibrils that trap an invading pathogen before the more differentiated adaptive immune system goes after it. In fact, several amyloid-forming proteins have antimicrobial properties (reviewed in Kagan et al., 2012). The similarities between AMPs and neuropeptides such as Aβ have led some scientists to hypothesize that neuropeptides might have anti-infective functions (Schluesener et al., 2012).

Previously, Tanzi’s group, together with the Moir laboratory at MGH, had proposed that Aβ counters microbes in vitro, and reported that amyloid-containing brain homogenates from people who died of Alzheimer’s have high antimicrobial activity (Apr 2009 conference news; Mar 2010 news). At the Zilkha meeting, Tanzi provided an update of the MGH scientists’ effort to explore the AMP hypothesis in a range of model systems.

They started small with human neuroglioma cultures that the researchers infected with Candida albicans, a type of yeast. Overexpressing Aβ in these cells protected them, doubling the number that remained uninfected, Tanzi told the audience. The researchers next infected Caenorhabditis elegans with Candida. In unprotected worms, the fungus grows to the point of breaking through their bellies. “It looks like the movie ‘Alien,’” Tanzi said. Half of the nematodes died within three days, but expressing Aβ in their muscle cells protected them; more than half were still alive after six days. Next Tanzi and Moir turned their sights on fruit flies, infecting Drosophila with Candida. Once again, wild-type flies perished within 50 days but those overexpressing Aß made it to 60.

Finally, the authors used Salmonella to infect the brains of four-week-old mice. For wild-type mice, this type of bacteria-derived meningitis means death within 60-72 days. Mice lacking the APP gene succumbed even faster, by 54 days, but 5xFAD mice survived to a maximum of 96 days, as if the excess Aβ protected them.

What blew the researchers’ minds, Tanzi said, was finding amyloid plaques when they examined the brains of the 5xFAD mice as soon as 48 hours after injecting Salmonella. Normally, these mice do not have plaques at the young age used in this study, but the infected animals did. And in the middle of each plaque was a Salmonella bacterium—the microbe seeded plaque formation around it. (Alzforum first reported this result in Apr 2015 conference news.)

This is what one would expect if the Aβ were an antimicrobial peptide. Moir and Tanzi have proposed a model they call the anti-microbial protection hypothesis, by which a subclinical or asymptomatic infection might seed amyloid formation, kicking off Alzheimer’s disease as a form of collateral damage. Older people might be more susceptible to this process as pathogens sneak into the brain through a blood-brain barrier compromised by age and adaptive immunity begins to wane (see Part 3 of this series).

Some other labs are starting to back up this hypothesis, reporting that Aβ inhibits both influenza virus and herpes simplex virus-1 (HSV1) (White et al., 2014; Bourgade et al., 2015; Bourgade et al., 2016). One implication, Tanzi told Alzforum, is that Aβ-lowering therapies should perhaps not be too potent lest recipients lose some innate protection against infectious agents. “Amyloid is not just junk,” he told the Zilkha audience. “We want to reduce it but not wipe it out.”

Microbiome Meets Aβ
While Tanzi considered invading microbes, Sisodia’s lab focused on the astounding 100 trillion or so bacteria that naturally inhabit the human gut. There have been previous hints that the natural microbiome could matter to the nervous system. For example, certain gut microbes can produce the neurotransmitter GABA, and germ-free mice have abnormally low expression of brain-derived neurotrophic factor in the hippocampus and cortex, along with abnormal behavior on tests of cognition and anxiety (reviewed in Bhattarcharjee and Lukiw, 2013). The brain’s resident immune cells, microglia, seem to depend on a healthy gut microbiome to mature properly (Jun 2015 news). And a sugar made by Bacteroides fragilis, a common intestinal denizen, protects mice from encephalomyelitis, a model condition for multiple sclerosis (Ochoa-Repáraz et al., 2010). 

Moreover, microbes themselves secrete amyloids, for example to help produce a sticky biofilm. These may contribute to neuropathology and AD, scientists posit (reviewed in Hill and Lukiw, 2015). One group found a higher level of infectious burden in people with AD, and proposed that bacterial infection could cause the immune system to confuse its own mitochondria or amyloids for invaders, and thus upregulate inflammation (Bu et al., 2014). 

Sisodia hypothesized that the makeup of the intestinal microbiome would influence neuroinflammation, and thus Aβ deposition, in the APPSWE/PSEN1dE9 mouse model of Alzheimer’s. The researchers treated mice from birth with a cocktail of eight antibiotics, assuming this would decrease the number of bacteria in their intestines.

That was not what happened, though. When the scientists checked the mice’s feces and cecum, they found plenty of microbes in both sources. “This came as a bit of a surprise,” Sisodia said. “We thought we would be wiping out the intestinal commensals.”

But were the bacteria that survived the antibiotic onslaught the same as those in untreated mice? Sisodia reasoned that antibiotic-resistant bacteria might have taken over. To gain a bird’s-eye view of the bacterial populations, the authors isolated their 16S rRNA and chopped it up with restriction enzymes to generate restriction fragment length polymorphisms (RFLPs). When they ran these out on a gel, clearly different RFLP patterns emerged between the antibiotic-treated and untreated mice. The populations must differ, Sisodia concluded. The researchers are now performing DNA sequencing to identify the particular bacteria in the mix.

Even if they did not manage to eliminate the microbiome, was just changing it sufficient to alter Aβ in the brain? Indeed it was. Plaque burden was halved in the antibiotic-treated mice. Insoluble Aβ42 levels also dropped, while the amount of soluble Aβ more than doubled.

Thus far in this new research project, the finding is robust only in male mice, Sisodia noted. While there was a trend toward reduced Aβ burden in the females, as well, it stayed below statistical significance with the nine to 10 mice used per group.

Presumably, the gut bacteria altered the peripheral immune response, which in turn changed the biology of the brain. To check immunity in the periphery, the researchers pooled serum from the antibiotic-treated mice and profiled it for cytokines, chemokines, and growth factors. They saw upregulation of several inflammatory mediators in the treated animals, some of which, such as CLC 11, can cross the blood-brain barrier. Sisodia speculated they might enter the brain and activate microglia.

Next, the researchers are raising germ-free 5xFAD mice to check how a missing microbiome affects amyloid deposition. They intend to repopulate those mice with a controlled bacterial population, to test which species or combinations affect amyloid.

Scientists at the Zilkha conference were quite interested in this microbial research direction. “It gives us a whole new perspective to the origin and understanding of AD,” said Philip Scheltens of the VU University Medical Center in Amsterdam. “We have to see how it plays out.”

“We have been scratching the surface of the microbiome, but it is really going to be important,” commented Maria Carrillo of the Alzheimer’s Association. She noted that some scientists have been pursuing a relationship between HSV1 and AD for well over a decade (Jamieson et al., 1991; Feb 2011 webinar). “Perhaps there is something to that,” said Carrillo.

Carrillo is not the only one who thinks so. In a recent editorial in the Journal of Alzheimer’s Disease, 33 AD researchers and clinicians argued that studies of microbes in AD have been neglected (Itzhaki et al., 2016). “We propose that further research on the role of infectious agents in AD causation, including prospective trials of antimicrobials therapy, is now justified,” the authors wrote.

The gut-brain axis is beginning to be explored in neurovascular research, as well. This month, a research collaboration including Costantino Iadecola at Weill Cornell Medical Center in New York reported that changing commensal bacteria with antibiotics reduced ischemic brain injury in mice via changes in T cells trafficking from the intestine to the brain after a stroke (Benakis et al., 2016). For more on neurovascular research in Alzheimer’s, see Part 3 of this series.—Amber Dance

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References

News Citations

1. Prague: Aβ Rehabilitated as an Antimicrobial Protein? 1 Apr 2009
2. Paper Alert: Aβ’s Day Job—Slayer of Microbes? 2 Mar 2010
3. Could Adaptive Immunity Set the Brakes on Amyloid? 23 Apr 2015
4. What’s Up With the Vasculature in Dementia? 10 May 2016
5. To Be Hale and Hearty, Brain Microglia Need a Healthy Gut 5 Jun 2015

Research Models Citations

1. 5xFAD (B6SJL)
2. APPswe/PSEN1dE9 (line 85)

Webinar Citations

1. Herpes Simplex and Alzheimer’s—Time to Think Again? 15 Apr 2011

Paper Citations

1. Kagan BL, Jang H, Capone R, Teran Arce F, Ramachandran S, Lal R, Nussinov R. Antimicrobial properties of amyloid peptides. Mol Pharm. 2012 Apr 2;9(4):708-17. Epub 2011 Nov 29 PubMed.
2. Schluesener HJ, Su Y, Ebrahimi A, Pouladsaz D. Antimicrobial peptides in the brain: neuropeptides and amyloid. Front Biosci (Schol Ed). 2012;4:1375-80. PubMed.
3. White MR, Kandel R, Tripathi S, Condon D, Qi L, Taubenberger J, Hartshorn KL. Alzheimer's associated β-amyloid protein inhibits influenza A virus and modulates viral interactions with phagocytes. PLoS One. 2014;9(7):e101364. Epub 2014 Jul 2 PubMed.
4. Bourgade K, Garneau H, Giroux G, Le Page AY, Bocti C, Dupuis G, Frost EH, Fülöp T Jr. β-Amyloid peptides display protective activity against the human Alzheimer's disease-associated herpes simplex virus-1. Biogerontology. 2015 Feb;16(1):85-98. Epub 2014 Nov 7 PubMed.
5. Bourgade K, Le Page A, Bocti C, Witkowski JM, Dupuis G, Frost EH, Fülöp T Jr. Protective Effect of Amyloid-β Peptides Against Herpes Simplex Virus-1 Infection in a Neuronal Cell Culture Model. J Alzheimers Dis. 2016;50(4):1227-41. PubMed.
6. Bhattacharjee S, Lukiw WJ. Alzheimer's disease and the microbiome. Front Cell Neurosci. 2013;7:153. PubMed.
7. Ochoa-Repáraz J, Mielcarz DW, Wang Y, Begum-Haque S, Dasgupta S, Kasper DL, Kasper LH. A polysaccharide from the human commensal Bacteroides fragilis protects against CNS demyelinating disease. Mucosal Immunol. 2010 Sep;3(5):487-95. Epub 2010 Jun 9 PubMed.
8. Hill JM, Lukiw WJ. Microbial-generated amyloids and Alzheimer's disease (AD). Front Aging Neurosci. 2015;7:9. Epub 2015 Feb 10 PubMed.
9. Bu XL, Yao XQ, Jiao SS, Zeng F, Liu YH, Xiang Y, Liang CR, Wang QH, Wang X, Cao HY, Yi X, Deng B, Liu CH, Xu J, Zhang LL, Gao CY, Xu ZQ, Zhang M, Wang L, Tan XL, Xu X, Zhou HD, Wang YJ. A study on the association between infectious burden and Alzheimer's disease. Eur J Neurol. 2014 Jun 9; PubMed.
10. Jamieson GA, Maitland NJ, Craske J, Wilcock GK, Itzhaki RF. Detection of herpes simplex virus type 1 DNA sequences in normal and Alzheimer's disease brain using polymerase chain reaction. Biochem Soc Trans. 1991 Apr;19(2):122S. PubMed.
11. Itzhaki RF, Lathe R, Balin BJ, Ball MJ, Bearer EL, Braak H, Bullido MJ, Carter C, Clerici M, Cosby SL, Del Tredici K, Field H, Fulop T, Grassi C, Griffin WS, Haas J, Hudson AP, Kamer AR, Kell DB, Licastro F, Letenneur L, Lövheim H, Mancuso R, Miklossy J, Otth C, Palamara AT, Perry G, Preston C, Pretorius E, Strandberg T, Tabet N, Taylor-Robinson SD, Whittum-Hudson JA. Microbes and Alzheimer's Disease. J Alzheimers Dis. 2016;51(4):979-84. PubMed.
12. Benakis C, Brea D, Caballero S, Faraco G, Moore J, Murphy M, Sita G, Racchumi G, Ling L, Pamer EG, Iadecola C, Anrather J. Commensal microbiota affects ischemic stroke outcome by regulating intestinal γδ T cells. Nat Med. 2016 May;22(5):516-23. Epub 2016 Mar 28 PubMed.

External Citations

1. TREM2 
2. CD33 

Further Reading

No Available Further Reading