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Cited by 372Alzheimer's disease research and treatmentsOral microbiology and periodontitis researchNeuroinflammation and Neurodegeneration Mechanisms

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Chronic oral application of a periodontal pathogen results in brain inflammation, neurodegeneration and amyloid beta production in wild type mice

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Ilievski, Vladimir, Zuchowska, Paulina K., Green, Stefan J., Toth, Peter T., Ragozzino, Michael E., Le, Khuong, Aljewari, Haider W., O’Brien-Simpson, Neil M., Reynolds, Eric C., Watanabe, Keiko·2018·PLOS ONE
DOI 10.1371/journal.pone.0204941PMC PMC6169940
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Abstract

BACKGROUND: The results from cross sectional and longitudinal studies show that periodontitis is closely associated with cognitive impairment (CI) and Alzhemer's Disease (AD). Further, studies using animal model of periodontitis and human post-mortem brain tissues from subjects with AD strongly suggest that a gram-negative periodontal pathogen, Porphyromonas gingivalis (Pg) and/or its product gingipain is/are translocated to the brain. However, neuropathology resulting from Pg oral application is not known. In this work, we tested the hypothesis that repeated exposure of wild type C57BL/6 mice to orally administered Pg results in neuroinflammation, neurodegeneration, microgliosis, astrogliosis and formation of intra- and extracellular amyloid plaque and neurofibrillary tangles (NFTs) which are pathognomonic signs of AD. METHODS: Experimental chronic periodontitis was induced in ten wild type 8-week old C57BL/6 WT mice by repeated oral application (MWF/week) of Pg/gingipain for 22 weeks (experimental group). Another 10 wild type 8-week old C57BL/6 mice received vehicle alone (control group) MWF per week for 22 weeks. Brain tissues were collected and the presence of Pg/gingipain was determined by immunofluorescence (IF) microscopy, confocal microscopy, and quantitative PCR (qPCR). The hippocampi were examined for the signs of neuropathology related to AD: TNFα, IL1β, and IL6 expression (neuroinflammation), NeuN and Fluoro Jade C staining (neurodegeneration) and amyloid beta1-42 (Aβ42) production and phosphorylation of tau protein at Ser396 were assessed by IF and confocal microscopy. Further, gene expression of amyloid precursor protein (APP), beta-site APP cleaving enzyme 1 (BACE1), a disintegrin and metalloproteinase domain-containing protein10 (ADAM10) for α-secretase and presenilin1 (PSEN1) for ɣ-secretase, and NeuN (rbFox3) were determined by RT-qPCR. Microgliosis and astrogliosis were also determined by IF microscopy. RESULTS: Pg/gingipain was detected in the hippocampi of mice in the experimental group by immunohistochemistry, confocal microscopy, and qPCR confirming the translocation of orally applied Pg to the brain. Pg/gingipain was localized intra-nuclearly and peri-nuclearly in microglia (Iba1+), astrocytes (GFAP+), neurons (NeuN+) and was evident extracellularly. Significantly greater levels of expression of IL6, TNFα and IL1β were evident in experimental as compared to control group (p<0.01, p<0.00001, p<0.00001 respectively). In addition, microgliosis and astrogliosis were evident in the experimental but not in control group (p <0.01, p<0.0001 respectively). Neurodegeneration was evident in the experimental group based on a fewer number of intact neuronal cells assessed by NeuN positivity and rbFOX3 gene expression, and there was a greater number of degenerating neurons in the hippocampi of experimental mice assessed by Fluoro Jade C positivity. APP and BACE1 gene expression were increased in experimental group compared with control group (p<0.05, p<0.001 respectively). PSEN1 gene expression was higher in experimental than control group but the difference was not statistically significant (p = 0.07). ADAM10 gene expression was significantly decreased in experimental group compared with control group (p<0.01). Extracellular Aβ42 was detected in the parenchyma in the experimental but not in the control group (p< 0.00001). Finally, phospho-Tau (Ser396) protein was detected and NFTs were evident in experimental but not in the control group (p<0.00001). CONCLUSIONS: This study is the first to show neurodegeneration and the formation of extracellular Aβ42 in young adult WT mice after repeated oral application of Pg. The neuropathological features observed in this study strongly suggest that low grade chronic periodontal pathogen infection can result in the development of neuropathology that is consistent with that of AD.

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Figures

Fig 1 Pg/gingipain is detected in the hippocampus of mice following oral application of Pg. (A) Immunofluorescence microscopy. Red: Pg, Blue: DAPI. (B) Images showing intra-, peri- and extra-cellularly localized Pg. (C) Number of Pg/gingipain detected per field (220μm X 160μm). N = 9 mice for experimental and N = 10 mice for control group. (D) Copy number of Pg 16S RNA genes detected in genomic DNA isolated from 5FFPE samples/mouse, N = 5 mice/group. Y- axis: number of Pg/gingipain per field for (C) and copy number of 16S rRNA genes from genomic DNA for (D). X-axis: C (control), E (experimental) group.
Fig 1 Pg/gingipain is detected in the hippocampus of mice following oral application of Pg. (A) Immunofluorescence microscopy. Red: Pg, Blue: DAPI. (B) Images showing intra-, peri- and extra-cellularly localized Pg. (C) Number of Pg/gingipain detected per field (220μm X 160μm). N = 9 mice for experimental and N = 10 mice for control group. (D) Copy number of Pg 16S RNA genes detected in genomic DNA isolated from 5FFPE samples/mouse, N = 5 mice/group. Y- axis: number of Pg/gingipain per field for (C) and copy number of 16S rRNA genes from genomic DNA for (D). X-axis: C (control), E (experimental) group.CC BY · PubMed Central
Fig 2 Pg/gingipain signals are detected peri- and intra-nuclearly by confocal microscopy. (A) CA1 region in 3-D. (B) Orthogonal analysis of 3-D image shown in A. Orthogonal projections a (green rectangle) and b (red rectangle) are side views of serial confocal sections of the same area. These images show intra-nuclear presence of Pg/gingipain.
Fig 2 Pg/gingipain signals are detected peri- and intra-nuclearly by confocal microscopy. (A) CA1 region in 3-D. (B) Orthogonal analysis of 3-D image shown in A. Orthogonal projections a (green rectangle) and b (red rectangle) are side views of serial confocal sections of the same area. These images show intra-nuclear presence of Pg/gingipain.CC BY · PubMed Central
Fig 3 Pg/gingipain is present intracellularly in astrocytes, microglia and neurons in the hippocampus of experimental mice. Green: astrocytes (top panels), microglia (middle panels), and neurons (bottom panels). Red: Pg/gingipain. Blue: DAPI. Representative of N = 4 (GFAP), 3 (Iba1) and 4 (NeuN) experimental mice.
Fig 3 Pg/gingipain is present intracellularly in astrocytes, microglia and neurons in the hippocampus of experimental mice. Green: astrocytes (top panels), microglia (middle panels), and neurons (bottom panels). Red: Pg/gingipain. Blue: DAPI. Representative of N = 4 (GFAP), 3 (Iba1) and 4 (NeuN) experimental mice.CC BY · PubMed Central
Fig 4 Proinflammatory cytokines IL6, IL1β and TNFα are evident in the hippocampus of experimental but not in control mice. Data from IF microscopy and RT-PCR. (A-F) Results from IF microscopy. IL6 expression in control (A) and experimental (B) mice, IL1β expression in control (C) and experimental (D) mice and TNFα expression in control (E) and experimental (F) mice. N = 3 mice/group. (G-I) Gene expression of cytokines was detected by RT-PCR (G, H, I). In all cases, there is significantly higher gene expression of all three cytokines in experimental compared with control group (G, H, I). Green: Iba1, Red: cytokines, Blue: DAPI. N = 5 mice/group.
Fig 4 Proinflammatory cytokines IL6, IL1β and TNFα are evident in the hippocampus of experimental but not in control mice. Data from IF microscopy and RT-PCR. (A-F) Results from IF microscopy. IL6 expression in control (A) and experimental (B) mice, IL1β expression in control (C) and experimental (D) mice and TNFα expression in control (E) and experimental (F) mice. N = 3 mice/group. (G-I) Gene expression of cytokines was detected by RT-PCR (G, H, I). In all cases, there is significantly higher gene expression of all three cytokines in experimental compared with control group (G, H, I). Green: Iba1, Red: cytokines, Blue: DAPI. N = 5 mice/group.CC BY · PubMed Central
Fig 5 The level of NeuN expression is lower in experimental mice compared with control mice (A-C). Images (A &B) are representative of NeuN detected by immunofluorescence microscopy and graph (D) is result from rbFOX3 gene expression analysis. (A) Hippocampus section from a control mouse. Insets show NeuN+ cells in CA1 and DG regions. (B) Hippocampus section from an experimental mouse. Insets show sparse NeuN+ cells in the CA1 and DG regions in experimental mouse. (C) The number of NeuN+ cells counted in CA1 and DG in representative 50μm X 100 μm rectangular areas. C: Control, E: Experimental. Green: NeuN, Blue: DAPI. (C) % NeuN+ cells in CA1 and DG regions (DAPI+ NeuN+/ DAPI+). Five fields per mouse counted. N = 10 for control and N = 9 for experimental group. (D) NeuN (rbFOX3) gene expression in control and experimental mice determined by RT-qPCR. N = 10 for control and N = 9 for experimental group.
Fig 5 The level of NeuN expression is lower in experimental mice compared with control mice (A-C). Images (A &B) are representative of NeuN detected by immunofluorescence microscopy and graph (D) is result from rbFOX3 gene expression analysis. (A) Hippocampus section from a control mouse. Insets show NeuN+ cells in CA1 and DG regions. (B) Hippocampus section from an experimental mouse. Insets show sparse NeuN+ cells in the CA1 and DG regions in experimental mouse. (C) The number of NeuN+ cells counted in CA1 and DG in representative 50μm X 100 μm rectangular areas. C: Control, E: Experimental. Green: NeuN, Blue: DAPI. (C) % NeuN+ cells in CA1 and DG regions (DAPI+ NeuN+/ DAPI+). Five fields per mouse counted. N = 10 for control and N = 9 for experimental group. (D) NeuN (rbFOX3) gene expression in control and experimental mice determined by RT-qPCR. N = 10 for control and N = 9 for experimental group.CC BY · PubMed Central
Fig 6 Neurodegeneration is evident in the hippocampus in the experimental group. (A) CA1 and DG regions from control mouse. There is no FJC staining in these regions. (B) CA1 and DG regions which show FJC+ staining in representative section from an experimental mouse. Green: FJC staining indicating degenerating neurons. Blue: DAPI. (C) FJC+ cells are present in the experimental but not in control mice. Positive cells were counted in 220μm x 175μm rectangular areas. Representative of N = 4 mice/group.
Fig 6 Neurodegeneration is evident in the hippocampus in the experimental group. (A) CA1 and DG regions from control mouse. There is no FJC staining in these regions. (B) CA1 and DG regions which show FJC+ staining in representative section from an experimental mouse. Green: FJC staining indicating degenerating neurons. Blue: DAPI. (C) FJC+ cells are present in the experimental but not in control mice. Positive cells were counted in 220μm x 175μm rectangular areas. Representative of N = 4 mice/group.CC BY · PubMed Central
Fig 7 Gene expression for APP, BACE1, PSEN1 and ADAM10 determined by RT-qPCR. Gene expression was significantly higher in experimental mice compared with control mice for APP (A) (p<0.05) and BACE1 (B) (p<0.001). There was no statistically significant difference in PSEN1 (C) expression between groups (p = 0.07). Gene expression for ADAM10 was significantly lower in the experimental compared with control group (D) (p<0.01). RNA was isolated from paraffin embedded sections and relative gene expression determined by qPCR. The results were normalized to β-actin gene expression. Y-axis: Relative gene expression, X-axis: C for control group, E for experimental group. N = 10 for control, N = 9 for experimental mice.
Fig 7 Gene expression for APP, BACE1, PSEN1 and ADAM10 determined by RT-qPCR. Gene expression was significantly higher in experimental mice compared with control mice for APP (A) (p<0.05) and BACE1 (B) (p<0.001). There was no statistically significant difference in PSEN1 (C) expression between groups (p = 0.07). Gene expression for ADAM10 was significantly lower in the experimental compared with control group (D) (p<0.01). RNA was isolated from paraffin embedded sections and relative gene expression determined by qPCR. The results were normalized to β-actin gene expression. Y-axis: Relative gene expression, X-axis: C for control group, E for experimental group. N = 10 for control, N = 9 for experimental mice.CC BY · PubMed Central
Fig 8 Aβ42 was detected in the hippocampus and cortex of experimental but not in control mice. Results from immunofluorescence microscopy. (A) Hippocampus of a control mouse showing no Aβ42, (B) hippocampus of an experimental mouse showing presence of Aβ42. Red: Aβ42, Blue: DAPI. Images in green frame are negative controls (isotype controls). (C) Aβ42 plaques counted per field (1.8mm X 1.3mm rectangle) which encompasses the entire right hemisphere of the hippocampus using 40X magnification. The same size rectangle was used to count plaque in the cortex (D-F). N = 10 for control and N = 9 for experimental groups. Images are 40X (top panels) and 100X insets for A and B.
Fig 8 Aβ42 was detected in the hippocampus and cortex of experimental but not in control mice. Results from immunofluorescence microscopy. (A) Hippocampus of a control mouse showing no Aβ42, (B) hippocampus of an experimental mouse showing presence of Aβ42. Red: Aβ42, Blue: DAPI. Images in green frame are negative controls (isotype controls). (C) Aβ42 plaques counted per field (1.8mm X 1.3mm rectangle) which encompasses the entire right hemisphere of the hippocampus using 40X magnification. The same size rectangle was used to count plaque in the cortex (D-F). N = 10 for control and N = 9 for experimental groups. Images are 40X (top panels) and 100X insets for A and B.CC BY · PubMed Central
Fig 9 Confocal microscopy of extracellular and intracellular Aβ42 around and in astrocytes in CA1 region of hippocampus and cortex of experimental mouse. (A) Confocal microscopy showing extracellular Aβ42 and intracellular Aβ42 in CA1 region of hippocampus. (B) and (C) are insets from (A). Red arrows in (C) point to intracellular Aβ42 (Yellow). Green: astrocytes (GFAP+), Red: Aβ42 detected by rabbit monoclonal antibody, Blue: DAPI. Images are representative of experimental mice (N = 4). (D-F) Images from confocal microscopy of cortex showing intracellular Aβ42 in 2-D (D), 3-D (E) and orthogonal analysis (F). (G-I) Images from confocal microscopy of hippocampus showing extracellular Aβ42 in 2-D (G), 3-D (H), and orthogonal analysis (I). Projections a and b are side views of serial confocal sections of the same area.
Fig 9 Confocal microscopy of extracellular and intracellular Aβ42 around and in astrocytes in CA1 region of hippocampus and cortex of experimental mouse. (A) Confocal microscopy showing extracellular Aβ42 and intracellular Aβ42 in CA1 region of hippocampus. (B) and (C) are insets from (A). Red arrows in (C) point to intracellular Aβ42 (Yellow). Green: astrocytes (GFAP+), Red: Aβ42 detected by rabbit monoclonal antibody, Blue: DAPI. Images are representative of experimental mice (N = 4). (D-F) Images from confocal microscopy of cortex showing intracellular Aβ42 in 2-D (D), 3-D (E) and orthogonal analysis (F). (G-I) Images from confocal microscopy of hippocampus showing extracellular Aβ42 in 2-D (G), 3-D (H), and orthogonal analysis (I). Projections a and b are side views of serial confocal sections of the same area.CC BY · PubMed Central
Fig 10 p-Tau (Ser 396) is detected by immunohistochemistry. NFT-like images are evident in experimental but not in control mice. Images are representative of N = 10 for control (A) and N = 9 for experimental (B) mice. (C) Number of p-Tau (Ser396) counted per field (1.8mm X 1.3mm rectangle) which encompasses the entire right hemisphere of the hippocampus using 40X magnification.
Fig 10 p-Tau (Ser 396) is detected by immunohistochemistry. NFT-like images are evident in experimental but not in control mice. Images are representative of N = 10 for control (A) and N = 9 for experimental (B) mice. (C) Number of p-Tau (Ser396) counted per field (1.8mm X 1.3mm rectangle) which encompasses the entire right hemisphere of the hippocampus using 40X magnification.CC BY · PubMed Central
Fig 11 Microgliosis is observed in both CA1 and DG regions of experimental but not control mice. Microglia (Iba1+ cells) in CA1 and DG regions in control (A) and experimental (B) mice. Blue: DAPI, Green: Iba1+ /microglia. (C) A significantly higher number of microglia are present in the experimental group compared with control group. Y-axis: number of microglia counted in a rectangular area (220μm X 160μm) in the hippocampus in 5 fields/sample. N = 5 mice/group. X-axis: C: control, E: experimental group.
Fig 11 Microgliosis is observed in both CA1 and DG regions of experimental but not control mice. Microglia (Iba1+ cells) in CA1 and DG regions in control (A) and experimental (B) mice. Blue: DAPI, Green: Iba1+ /microglia. (C) A significantly higher number of microglia are present in the experimental group compared with control group. Y-axis: number of microglia counted in a rectangular area (220μm X 160μm) in the hippocampus in 5 fields/sample. N = 5 mice/group. X-axis: C: control, E: experimental group.CC BY · PubMed Central
Fig 12 Astrogliosis was evident in the hippocampus of experimental but not in control mice. Astrocytes (GFAP+ cells) in CA1 and DG regions in control (A) and experimental (B) mice. Green: GFAP+ cells, Blue: DAPI. (C) A significantly higher number of astrocytes are present in the experimental group compared with control group. Y-axis: number of astrocytes counted in a rectangular area (990μm X 680μm) in the hippocampus in 5 fields/sample. N = 4/group. X-axis: C for control, E for experimental group.
Fig 12 Astrogliosis was evident in the hippocampus of experimental but not in control mice. Astrocytes (GFAP+ cells) in CA1 and DG regions in control (A) and experimental (B) mice. Green: GFAP+ cells, Blue: DAPI. (C) A significantly higher number of astrocytes are present in the experimental group compared with control group. Y-axis: number of astrocytes counted in a rectangular area (990μm X 680μm) in the hippocampus in 5 fields/sample. N = 4/group. X-axis: C for control, E for experimental group.CC BY · PubMed Central

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  1. CS Little, 2014, Detection of bacterial antigens and Alzheimer's disease-like pathology in the central nervous system of BALB/c mice following intranasal infection with a laboratory isolate of Chlamydia pneumoniae, Front Aging Neurosci, 6, 304, 10.3389/fnagi.2014.00304 · DOI · Paper page

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Metadata

Authors
Ilievski, V., Zuchowska, P. K., Green, S. J., Toth, P. T., Ragozzino, M. E., Le, K., Aljewari, H. W., O’Brien-Simpson, N. M., Reynolds, E. C., & Watanabe, K.
Affiliations
  • University of Illinois Chicago (US)
  • The University of Melbourne (AU)
Corresponding author
Watanabe, Keiko
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2018-10-03
Volume
13
Issue
10
Pages
e0204941
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0204941
PMC
PMC6169940
OpenAlex
W2895374072
Cited by
372
References
74
Citations by year
2026: 40 · 2025: 55 · 2024: 53 · 2023: 48 · 2022: 42 · 2021: 61 · 2020: 51 · 2019: 21 · 2018: 1
FWCI
22.97
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • Minerva Access (University of Melbourne) (submittedVersion, cc-by)
Funders
National Institutes of Health (R01DE021405)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Health Sciences › Dentistry › Periodontics › Oral microbiology and periodontitis research
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
periodontitis, Porphyromonas gingivalis, gingipains, Alzheimer's disease, neuroinflammation, neurodegeneration, amyloid-beta, tau phosphorylation, microglial activation, astrocyte activation, amyloid plaques, neurofibrillary tangles, hippocampus, APP processing, BACE1, ADAM10
MeSH terms
ADAM10 Protein, Administration, Oral, Alzheimer Disease, Animals, Astrocytes, Cell Count, Cross-Sectional Studies, DNA-Binding Proteins, Encephalitis, Frontal Lobe, Gene Expression Regulation, Hippocampus, Male, Mice, Inbred C57BL, Nerve Tissue Proteins, Neurons, Nuclear Proteins, Peptide Fragments, Periodontitis, Amyloid beta-Peptides, Aspartic Acid Endopeptidases, tau Proteins, Porphyromonas gingivalis, Microglia, Intracellular Space, Mice, Presenilin-1, Amyloid Precursor Protein Secretases, Cognitive Dysfunction

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