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, 87 (22), 2324-2332

Gram-negative Bacterial Molecules Associate With Alzheimer Disease Pathology

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Gram-negative Bacterial Molecules Associate With Alzheimer Disease Pathology

Xinhua Zhan et al. Neurology.

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 < 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 Aβ1-40/42 in amyloid plaques and with Aβ1-40/42 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 Aβ1-40/42 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 Aβ and resemble amyloid-like plaques.

Figures

Figure 1
Figure 1. Western blot analysis of Escherichia coli K99 pili protein and Gram-negative bacterial lipopolysaccharide (LPS) in human brains
(A) Western blot analysis of E coli K99 pili protein in gray matter (GM). E coli K99 was detected in 9 out of 13 Alzheimer disease (AD) cases and 1 out of 10 control (C) cases. (B) Western blot analysis of E coli K99 pili protein in white matter (WM). E coli K99 was detected in 10 out of 13 AD cases and 4 out of 10 control cases. (C) Quantification of E coli K99 protein. E coli K99 protein levels in AD brains were greater than in control aged brains in both GM (*p < 0.05) and WM (**p < 0.01). (D) Western blot analysis of Gram-negative bacterial LPS. A total of 3/3 AD GM, 3/3 AD WM, and 0/3 control GM showed LPS. Error bars are standard errors of the mean. β-actin was used as a loading control.
Figure 2
Figure 2. Immunofluorescence of lipopolysaccharide (LPS) in human brains
Immunocytochemistry for LPS showed staining in both control (A, C) and Alzheimer disease (AD) brains (B, D) in gray matter (GM) (A, B) and in periventricular white matter (PVWM, C, D). LPS colocalized with 4',6-diamidino-2-phenylindole (DAPI)–stained nuclei in control GM (E) and WM (G) including ependymal cells (G, arrowheads). The patterns of control LPS staining in the DAPI-stained nuclei varied (E; yellow, green, and white arrowheads). In AD GM, there were large foci of LPS staining that appeared to be plaques in cortex (Ctx) (B, F; white arrow), and other LPS-stained structures that appeared to be the size of nuclei in GM (B, F; yellow arrowheads). LPS staining was detected in control (C, G) and AD (D, H) WM. DAPI staining of nuclei was decreased in AD Ctx (F) and in WM (H) including ventricular ependymal cells (H, white arrowheads), likely indicating cell loss. LPS was detected in all 11 AD brains and all 7 control brains by immunofluorescence. Bar = 25 μm.
Figure 3
Figure 3. Immunofluorescence of Escherichia coli K99 pili protein in human brains
E coli K99 staining in control cortex (Ctx) was punctate and not associated with 4',6-diamidino-2-phenylindole (DAPI)–stained nuclei (A). In control white matter, K99 staining appeared to be associated with fiber tracts (C). In Alzheimer disease (AD) gray matter (GM), E coli K99 pili protein was often found in pyramidal cells in the cytoplasm and not the nucleus (B, arrowheads). There was intense E coli K99 pili protein staining in AD periventricular white matter (PVWM) (D) with loss of normal DAPI-stained ependymal cells in AD brains (D, arrowheads) compared to controls (C, arrowheads). E coli K99 pili protein was detected in all 11 AD brains and all 7 control brains by immunofluorescence. Bar = 25 μm.
Figure 4
Figure 4. Association of lipopolysaccharide (LPS) and Escherichia coli K99 pili protein with amyloid plaques in Alzheimer disease (AD) brains
There were several different patterns of colocalization of LPS and Aβ1-40/42 in AD brains. There were clusters of predominantly LPS particles that colocalized with Aβ1-40/42 (A). There were Aβ1-40/42 deposits that colocalized with LPS and were surrounded by LPS (B, C). Finally, there were confluent Aβ1-40/42-stained amyloid plaques that had scattered LPS particles within them (D). These LPS results contrasted with E coli K99 pili protein, which often surrounded small Aβ1-40/42-stained amyloid plaques (E–G). For larger amyloid plaques (diameter >50 μm), E coli K99 was usually absent (H). Bar = 25 μm.
Figure 5
Figure 5. Association of lipopolysaccharide (LPS) and Escherichia coli K99 with blood vessels
LPS and E coli K99 were also found in vessels of human brains. In control brains, both LPS (A) and E coli K99 pili protein (C) were localized in blood vessels that did not stain for Aβ1-40/42 (A, C). LPS, E coli K99 pili protein, and Aβ1-40/42 were localized in vessels of Alzheimer disease (AD) brains (B, D). Aβ1-40/42 colocalized (yellow staining) with LPS in vessels in AD brains (B). Aβ1-40/42 was frequently sandwiched by but did not colocalize with E coli K99 pili protein (D, arrowheads). Bar = 25 μm.

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