Bocharova O, Pandit NP, Molesworth K, Fisher A, Mychko O, Makarava N, Baskakov IV. Alzheimer's disease-associated β-amyloid does not protect against herpes simplex virus 1 infection in the mouse brain. J Biol Chem. 2021 Jul;297(1):100845. Epub 2021 May 28 PubMed.

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  1. Bocharova et al. present an interesting perspective on the role of Aβ as an antimicrobial peptide. Rob Moir and colleagues had reported earlier that Aβ has antimicrobial activity in vitro against bacteria and yeast (Soscia et al., 2010), agglutinates Candida albicans cells in vitro (Kumar et al., 2016), and that transgenic Caenorhabditis elegans nematodes expressing human Aβ1–42 show increased survival following challenge with C. albicans (Kumar et al., 2016). In this same paper, they reported that transgenic 5xFAD mice overexpressing human Aβ were differentially protected against challenge with the bacterium Salmonella typhimurium (P = <0.0001). A fivefold reduction of brain bacterial load was seen in the transgenic mice. In a later paper (Eimer et al., 2018) the same team showed that 5xFAD mice (5–6 weeks, n = 14, all female, heterozygous BL6/SJL background) were protected (P = 0.045) against challenge with HSV-1 (strain unspecified; 108/9 PFU of virus), but protection was partial, and all challenged animals eventually succumbed irrespective of genotype.

    Bocharova et al. attempted to repeat these experiments, also using 5xFAD mice (5–6 weeks) on a similar genetic background, and two different strains of HSV-1 (17syn+ and McKrae). Although they state that 5xFAD mice were not differentially protected, mice challenged with 104 PFU of HSV-1 McKrae (the highest dose tested for this virus) did in fact show partial protection, and two of seven transgenic mice survived to 350 h post-infection, whereas all control mice (n = 8) died (P = 0.075). Thus, this trends toward confirming the previous report (Eimer et al., 2018), and would probably have achieved P = <0.05 if they had used more animals.

    However, these are tricky experiments: In addition to the need for biological containment of live viruses, transgenic mice are finicky and expensive (hence the low numbers used). Mice are not a natural host for HSV-1, and different mouse strains show very different susceptibilities to HSV-1. BL6 mice are said to be highly resistant whereas SJL mice are sensitive (oral infection with 2 × 105 PFU) (Kastrukoff et al., 2012); the mixed BL6 and SJL genetic background therefore adds uncertainty. Furthermore, Bocharova et al. used “random” male and female mice, whereas Eimer et al. only used females; female mice are reported to be 27-fold more susceptible to HSV-1 than male mice (Geurs et al., 2012). In short, individual mice in the experiment of Bocharova et al. could have had different levels of innate susceptibility, potentially confounding interpretation.

    Bocharova et al. also reported no correlation between viral load and genotype, but only investigated cortex, which does not express HSV-1 receptors (Lathe and Haas, 2017), where virions presumably arrived by simple diffusion, not through local replication). They also found no co-localization of HSV-1 with Aβ; this might be expected because Aβ expression in 5xFAD mice is predominantly constitutive, whereas in natural settings it is induced by infection/inflammation. Overall, although there are some potential shortcomings, this is a welcome and conscientious study. Indeed, the authors point out that their work does not refute the viral hypothesis of AD.

    But are we looking at the right microbe? These studies raise an obvious question: What is the natural target for Aβ antimicrobial action? Antimicrobial proteins differ widely in their activity against different pathogens, and the marginal in vivo protection against HSV-1 in 5xFAD mice (Eimer et al., 2018; and Bocharova et al.) contrasts with the reported strong protective effects against C. albicans and Salmonella. In vitro, Aβ reduced HSV-1 infectivity by only around 30 percent (Eimer et al., 2018; see also Bourgade et al., 2016), but inactivated >90 percent of Enterococcus faecalis and was even better against Candida albicans (Soscia et al., 2010). This could tie in with reports of diverse microbial infections of the brain (Lathe and St. Clair, 2020). It would certainly be of interest to explore Aβ’s antimicrobial activity against a much wider range of microbes, including the spirochete Treponema pallidum and the protozoan Toxoplasma gondii, both of which have been implicated in brain disease.

    References:

    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.

    Eimer WA, Vijaya Kumar DK, Navalpur Shanmugam NK, Rodriguez AS, Mitchell T, Washicosky KJ, György B, Breakefield XO, Tanzi RE, Moir RD. Alzheimer's Disease-Associated β-Amyloid Is Rapidly Seeded by Herpesviridae to Protect against Brain Infection. Neuron. 2018 Jul 11;99(1):56-63.e3. PubMed.

    Geurs TL, Hill EB, Lippold DM, French AR. Sex differences in murine susceptibility to systemic viral infections. J Autoimmun. 2012 May;38(2-3):J245-53. Epub 2011 Dec 29 PubMed.

    Kastrukoff LF, Lau AS, Thomas EE. The effect of mouse strain on herpes simplex virus type 1 (HSV-1) infection of the central nervous system (CNS). Herpesviridae. 2012 Mar 26;3:4. PubMed.

    Kumar DK, Choi SH, Washicosky KJ, Eimer WA, Tucker S, Ghofrani J, Lefkowitz A, McColl G, Goldstein LE, Tanzi RE, Moir RD. Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer's disease. Sci Transl Med. 2016 May 25;8(340):340ra72. PubMed.

    Lathe R, Haas JG. Distribution of cellular HSV-1 receptor expression in human brain. J Neurovirol. 2017 Jun;23(3):376-384. Epub 2016 Dec 15 PubMed.

    Lathe R, St Clair D. From conifers to cognition: Microbes, brain and behavior. Genes Brain Behav. 2020 Nov;19(8):e12680. Epub 2020 Jul 9 PubMed.

    Soscia SJ, Kirby JE, Washicosky KJ, Tucker SM, Ingelsson M, Hyman B, Burton MA, Goldstein LE, Duong S, Tanzi RE, Moir RD. The Alzheimer's disease-associated amyloid beta-protein is an antimicrobial peptide. PLoS One. 2010 Mar 3;5(3):e9505. PubMed.

    View all comments by Richard Lathe

  2. Bocharova et al. tested 5XFAD mice in an attempt to replicate our results demonstrating a protective role of Aβ against HSV1 (Eimer et al., 2018). Bocharova et al. present data intended to suggest the contrary; however, we do not believe their data constitute sufficient contradictory evidence against Aβ in its role as an antimicrobial peptide, or its protective effects against HSV1.

    In both our paper and Bocharova et al., survival experiments of 5- to 6-week-old 5xFAD mice were carried out. In contrast to our study, Bocharova et al. claimed no significant protective effects against HSV1 in 5XFAD versus wild-type mice. Bocharova et al. began to observe trends toward enhanced survival at the highest viral load used, i.e., 1x104 PFU. But then, they did not test higher viral loads to replicate the viral load used in our study, which was 1x108 PFU—10,000 times higher. Perhaps, had the Bocharova et al. survival studies extended to higher doses of virus, they also would also have observed a significant degree of protection against HSV1 infection by Aβ, as we reported.

    Bocharova et al.’s results are based on immunofluorescent analysis of both young and old 5XFAD mice infected with HSV1, and their inability to observe HSV1 co-localization with Aβ plaques. A significant difference between our study and Bocharova et al. is how HSV1 was visualized. We presented images with HSV1 labeled with a red-fluorescent protein attached to the VP-26 capsid protein, leaving no doubt about its presence or absence. Bocharova et al. used antibodies targeting surface glycoproteins gD and gH. While in our previous studies we showed that Aβ can bind gD and gH, importantly, Bocharova et al. carried out no controls to test whether Aβ interferes with antibody binding, which our unpublished preliminary data suggests. Bocharova et al. cannot properly justify their conclusion that there is no overlap of HSV1 and Aβ in plaques in the absence of this essential control.

    Other data presented by Bocharova et al. actually do provide potential support for Aβ’s protective role as an antimicrobial peptide. Immunofluorescent analysis in both Fig. 4 (of young mice) and Fig. 5 (of old mice) presented reduced or absent HSV1 signal in the high Aβ production areas where the Thy1 promoter is most prominent. This could be interpreted as a protective effect being localized to high amyloid concentration areas in the brain.

    Fig. S3 revealed reduced Aβ plaque load in HSV1-challenged mice that survived versus unchallenged 5XFAD littermates. Bocharova et al. interpreted this as HSV-1 clearance without triggering Aβ deposition. We interpret this to be due to Aβ’s antimicrobial binding to HSV1, thereby reducing free Aβ levels. This would subsequently delay the formation of amyloid plaques until sufficient concentration for aggregation was once again achieved. Finally, aged 5XFAD mice presented a trend of reduced HSV1 copy number. While not significant, the trend is evident. Perhaps, if Bocharova et al. had increased the number of mice used, the high variance in their results could have been avoided.

    Both the role of Aβ as an antimicrobial peptide and the potential role for microbial pathogens in AD are emerging hypotheses in the field right now. Rigorous analysis of the existing work and continued expansion of data is needed to move the field forward in these new directions. In the absence of any in vitro testing of the antimicrobial properties of Aβ, and, in our opinion, the inconclusive in vivo data in 5XFAD mouse, we do not believe Bocharova et al. provide the necessary evidence to refute our conclusions in Eimer et al., 2018. While we disagree with the overall conclusions in Bocharova et al., we do welcome the continued analysis of our hypothesis.

    References:

    Eimer WA, Vijaya Kumar DK, Navalpur Shanmugam NK, Rodriguez AS, Mitchell T, Washicosky KJ, György B, Breakefield XO, Tanzi RE, Moir RD. Alzheimer's Disease-Associated β-Amyloid Is Rapidly Seeded by Herpesviridae to Protect against Brain Infection. Neuron. 2018 Jul 11;99(1):56-63.e3. PubMed.

    View all comments by Rudy Tanzi

  3. To date, I’m not convinced by any of the experimental or clinical evidence that a conventional virus underlies the pathogenesis of AD. Latent or temperate conventional viral activity may, however, play a role in the relatively common phenocopy of AD, marked by TDP43. This condition, variously known as hippocampal sclerosis, frontotemporal dementia, Pick’s disease, FTD/ALS spectrum, FTLD, LATE (and variants thereof) has many of the hallmarks of a latent herpes virus with its topographic propensity to involve the limbic system, possibly associated with a virus-initiated subclinical auto-inflammatory/auto-immune process. The key may lie in the property of TDP43 localizing/binding to foreign nucleic acids in the cytoplasm.

    Is AD an unconventional, slow “virus,” i.e., an autocatalytic prion disease of Aβ amyloid? Maybe, but the evidence is hard to come by. Recent cases of Aβ angiopathy linked to childhood neurosurgery and dura mater grafts are beginning to raise awareness of this possibility. The advent of reliable Aβ biomarkers should help shed some light on this.

    Do tau and α-synuclein also share this autocatalytic activity? Convincing quantitative evidence for amplification of inoculated “seeds” remains lacking.

    View all comments by Colin Masters

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