Gram-negative bacterial molecules associate with Alzheimer disease pathology
Authors: Lee-Way Jin
Brett Phinney
Boryana Stamova
Frank R Sharp
Charles DeCarli
Xinhua Zhan
Journal: Neurology (2016)
Discipline: NEUROSCIENCE & BEHAVIOR
Keywords: Gram-negative bacteria; Alzheimer disease; neuropathology; E coli K99; LPS
Affiliations: From the Department of Neurology (X.Z., B.S., C.D., F.R.S.), MIND Institute (X.Z., B.S., F.R.S.), Alzheimer's Disease Center (L.-W.J., C.D.), Department of Pathology (L.-W.J.), and Proteomics Core Facility, Genome Center (B.P.), University of California at Davis, Sacramento. ., From the Department of Neurology (X.Z., B.S., C.D., F.R.S.), MIND Institute (X.Z., B.S., F.R.S.), Alzheimer's Disease Center (L.-W.J., C.D.), Department of Pathology (L.-W.J.), and Proteomics Core Facility, Genome Center (B.P.), University of California at Davis, Sacramento.

Abstract:  Objective: We determined whether Gram-negative bacterial molecules are associated with Alzheimer disease (AD) neuropathology given that previous studies demonstrate Gram-negative Escherichia coli bacteria can form extracellular amyloid and Gram-negative bacteria have been reported as the predominant bacteria found in normal human brains. Methods: Brain samples from gray and white matter were studied from patients with AD (n = 24) and age-matched controls (n = 18). Lipopolysaccharide (LPS) and E coli K99 pili protein were evaluated by Western blots and immunocytochemistry. Human brain samples were assessed for E coli DNA followed by DNA sequencing. Results: LPS and E coli K99 were detected immunocytochemically in brain parenchyma and vessels in all AD and control brains. K99 levels measured using Western blots were greater in AD compared to control brains (p &lt; 0.01) and K99 was localized to neuron-like cells in AD but not control brains. LPS levels were also greater in AD compared to control brain. LPS colocalized with AŒ≤1-40/42 in amyloid plaques and with AŒ≤1-40/42 around vessels in AD brains. DNA sequencing confirmed E coli DNA in human control and AD brains. Conclusions: E coli K99 and LPS levels were greater in AD compared to control brains. LPS colocalized with AŒ≤1-40/42 in amyloid plaques and around vessels in AD brain. The data show that Gram-negative bacterial molecules are associated with AD neuropathology. They are consistent with our LPS-ischemia-hypoxia rat model that produces myelin aggregates that colocalize with AŒ≤ and resemble amyloid-like plaques. 

Retracted: No

PubPeer comments:
 I was surprised by the appearance of LPS on the Western blot in Figure 1. It shows a strong single immunoreactive band at approximately 35 kDa in the AD patient brains. As far as I understand, LPS shows up on Western blots as a ladder, and not a single band. It was also surprising that the K99 antigen also showed up as a single immunoreactive band at approximately 35 kDa (same size as the LPS band). This antigen seems to be about 18 kDa on other groups Westerns. This would lead me to speculate that there is something at 35 kDa in these brains that is reacting non-specifically. The Abcam LPS antibody listed is suggested for IF and ELISA on the manufacturer s website (not Western) and the Ray Biotech one does not seem to exist. The K99 antigen is also suggested for ELISA not Western.  , 
 Responses: We did realize LPS shows up on Western blots as different patterns of ladders depends on the strains. As far as I know, those LPS are purified lipopolysaccharides without interactions with biological system. LPS is an endotoxin which induces a strong immune response from normal animal and human. The LPS patterns presented in the brain might alter after the involvements of peripheral and CNS innate immune responses. We hypothesize that some cytokines or other inflammatory molecules are interacted with LPS, which forms a complex with a major MW around 37 kDa though you still can tell some ladder-like thin bands around 37 kDa in brains when the band are not too intensive such as AD3 of white matter. In addition, when used another antibody against LPS derived from different strain of E. coli (O83:H1) for Western blot, we detected the LPS patterns more like ladders than them in the Figure 1. The most intensive band among the ladders is still around 37 kDa:  Similar to LPS, ki99 bands might be associated with the immune complex as well. We performed immunofluorescence (IF) of LPS as suggested by the manufacturer. We tested the specificity of LPS using two methods: Immunoabsorption specificity controls by 1) Western blot (Figure e-2) and 2) by IF (Figure e-3). After immunoabsorption of LPS antibody by LPS, the 37 kDa band is disappeared and IF staining is disappeared, which suggests LPS antibody used in the study is specific to LPS.   We did purchase one type of LPS antibody from Ray Biotech (MD-05-0148) and used it in our study. However, it does not show on the website anymore since it was discontinued, which is confirmed by technical support (email: techsupport@raybiotech.com; phone: (888) 494-8555). We still have the vial of the product though:  ,