Porphyromonas gingivalisin Alzheimer‚Äôs disease brains: Evidence for disease causation and treatment with small-molecule inhibitors
Authors: Mai Nguyen
Leslie J Holsinger
Hatice Hasturk
Samer Kaba
Eric C Reynolds
Jan Potempa
Alexander Lee
Casey Lynch
Agata Marczyk
Richard L M Faull
Barbara Potempa
Piotr Mydel
Florian Ermini
Malgorzata Benedyk
Stephen S Dominy
Christina Griffin
Annelie Hellvard
Karina Adamowicz
Ursula Haditsch
Mike Dragunow
Maurice A Curtis
Mark I Ryder
Glenn D Walker
Shirin Arastu-Kapur
Andrei Konradi
Debasish Raha
Journal: Science Advances (2019)
Discipline: Multidisciplinary
Keywords: Porphyromonas gingivalis; Alzheimer's disease; gingipains; tau; AŒ≤1-42
Affiliations: Cortexyme, Inc., 269 East Grand Ave., South San Francisco, CA, USA., Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland., Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland., Division of Periodontology, Department of Orofacial Sciences, University of California, San Francisco, San Francisco, CA, USA., Department of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, Louisville, KY, USA., Broegelman Research Laboratory, Department of Clinical Science, University of Bergen, Bergen, Norway., The Forsyth Institute, Cambridge, MA, USA., Harvard University School of Dental Medicine, Boston, MA, USA., Cooperative Research Centre for Oral Health Science, Melbourne Dental School and the Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Melbourne, Victoria, Australia., Department of Anatomy with Radiology, Centre for Brain Research and NeuroValida, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand., Centre for Brain Research and NeuroValida, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand., Department of Anatomy and Medical Imaging, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand., Department of Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.

Abstract:  Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of AŒ≤1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked AŒ≤1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease. 

Retracted: No

PubPeer comments:
   This paper has several issues, including lacking controls for antibody specificity, equal protein loading, and overstated misleading data (a few of these are summarized below). Perhaps most concerning however are these images showing duplicated figures (Figure 4C and S4B, showing clearly that the control lanes have been spliced on), and also a spliced gel in Figure 8C. At best, this is very sloppy science, but at worst... I m perplexed as to how this paper was one of the major pieces of evidence to secure the backing company (Cortexyme) $76 million in Series B funding, let alone pass peer review in Science Advances? 1- Figure 3A and B: (Probable) Non-specific bands for Kgp, present in control and AD samples, blots lacking appropriate Kgp KO controls as well as loading controls. 2- Figure 5A: The Tau-5 antibody detects specific bands from 40-50 kD from SH-SY5Y cell lysates (Source: https: www.sigmaaldrich.com catalog product mm 577801?lang=en region=US). However, only bands higher than 75kD are shown. 3- Figure 7C: P. gingivalis-infected mice have brain levels of AB42 around 1pg mg tissue, which the authors claim is enough to cause AD-like symptoms (mock infections had 0.75pg mg) and is reversible by their gingipain inhibitors. Other mouse models known to develop AD-like symptoms have brain AB42 levels of 40+pg mg tissue (see Fig. 1 from PMC5789736). 4- Figure 4A and B: No correlation between saliva P. gingivalis levels and CSF P. gingivalis levels in AD patients. One would reason that a larger presence of oral P. gingivalis would lead to more in the nervous system, according to their hypothesis. 5- Figure 8E. Ex-vivo cultured plaques taken from periodontitis patients barely (if at all) express the Kgp gingipain (CB2 and CB3). , 
 The anonymous poster, Phaeoceros Evanidus makes a number of scientifically muddled complaints many of which are addressed in the manuscript. u00a0 None of the complaints are relevant to the conclusions ofu00a0the paper and the gingipainu00a0hypothesis. Re 3A and B: The Methods section, page 13, describes that the gingipain antibodies, including anti-Kgp antibody CAB102, were validated for specificity in human brain. As shown, Kgp is known to be present at multiple molecular weights under physiological conditions consistent with those demonstrated. It doesn t make scientific sense to ask for loading controls for an immunoprecipitation. Figure 4A and B: There is no a priori assumption that saliva P. gingivalis DNA and CSF P. gingivalis DNA should correlate, data is simply presented. Though it might be possible to find a correlation between P. gingivalis DNA in the saliva and P. gingivalis DNA in the CSF, an infection of P. gingivalis in the brain could also become compartmentalized and evolve independently of the original peripheral source of the infection over time and that is what the data shows. Figure 4C and supplemental Fig 4B: As noted, supplemental figure 4B does illustrate the break between the AD samples and the negative positive control lanes. While not ideal, the methods describe that this assay used multiple rounds of amplification of extremely small levels of cell free DNA so its reasonable that care would need to be taken to separate positive controls from the samples to avoid false positives. Figure 5A: In the paper, page 5, it specifically states that u201cTo determine whether gingipains cleave tau in a cell-based system, we used SH-SY5Y cells that express high-molecular weight forms of tau (50)u201d. Reference 50 in the paper is: Uberti et al., Characterization of tau proteins in human neuroblastoma SH-SY5Y cell line. Neurosci Lett. 235, 149-153 (1997). In the Uberti paper it is made clear that undifferentiated SH-SY5Y cells, which were in the experiments depicted in Fig. 5A, express high-molecular weight tau. 7C: The entire premise of the paper is that AB42 is an antimicrobial host response to P. gingivalis infection, and gingipains, not AB42, drive neurodegeneration, and there are multiple lines of evidence presented to support this. Therefore, the comparison of the amount of AB42 generated in the brain of a wild-type mouse in response to P. gingivalis infection to the amount of AB42 in the brain of an artificial transgenic-mouse designed to overproduce AB42 is irrelevant. Figure 8c: This is a minor figure to establish the Kgp-specificity of the probe used to look at Kgp activity in subsequent figures. The third lane could have and should have been left out entirely without impacting the interpretation of the specificity of the probe and conclusion of subsequent figures. Figure 8E. Phaeoceros has incorrectly stated the method used in the experiment depicted in Figure 8E. The band strength will be based on the sensitivity of the fluorescent probe and the differences between the different subjects. u00a0Some subjects have strong Kgp activity, and some have very little, and the small-molecule COR388 clearly inhibits Kgp activity, which was the goal of the experiment. Unclear what the complaint is or why certain levels of Kgp activity would be assumed. The figure appropriately reports what was observed. ,