Gingipain Causes Tau Tangle Formation in Alzheimer’s Disease

Brains via Regulation of TREM-1

Yiyi Zhu

Department of life sciences, Imperial College London, London, U.K.

Keywords: STREM-1, Gingipain, Tau, Alzheimer’s Disease.

Abstract: Tau phosphorylation is widely believed as an indicator of Alzheimer’s Disease (AD). Recent evidence

suggests that the periodontal pathogen, Porphyromonas gingivalis, is associated with AD development by

increasing the level of tau protein in the brain. Gingipain, the major virulence factor of the bacteria, has been

described to be involved in the cleavage of the Triggering Receptor Expressed on Myeloid cells 1 (TREM-

1). The soluble TREM-1 (sTREM-1) generated was shown to result in the activation of tau phosphorylation.

This work aims to uncover the underlying mechanism by which P. gingivalis causes AD by studying the

relationship between the level of gingipain, TREM-1 and tau in the brain of mice infected with P. gingivalis.

Through this study, a novel mechanism of AD formation may be proposed and can be exploited to generate

therapies against the disease.

1 INTRODUCTION

Alzheimer’s disease (AD) is one of the leading causes

of dementia worldwide. The widely accepted cause of

AD is the loss of neurons caused by the formation of

neurofibrillary tangles from tau phosphorylation

(Niikura, Tajima, Kita 2006, Ryder 2020). However,

the mechanism behind this process is not clear, and

recent studies have shown that the gram-negative

bacteria Porphyromonas gingivalis responsible for

periodontal infections has been detected in the brain

of AD patients, which suggest a role of the bacteria in

the pathogenesis of the disease (Dominy et al 2019).

The major virulence factor produced by the

bacteria is a family of conserved proteases called

gingipain n (Dominy et al 2019). This family of

protease consists of lysine-gingipain (Kgp), arginine-

gingipain A (RgpA), and arginine-gingipain B

(RgpB), which were found in the brain of 90% of the

AD patients (Haditsch 2020). These proteases are

localized to the hippocampus and result in the

secretion of pro-inflammatory cytokines that cause

damage to the brain (Ilievski et al 2018).

Experiments using human polymorphonuclear

neutrophils have demonstrated a role of Rgp in the

shredding of the triggering receptor expressed on

myeloid cells 1 (TREM1), which level is found to be

higher in AD patients (Sao et al 2018). TREM1

enhances cellular response by the recruitment of more

inflammatory cells (Bouchon, Dietrich, Colonna

2000). A study using human plasma demonstrated a

positive relationship between the level of sTREM1

and tau protein in AD patients, thus confirming the

role of sTREM1 in AD development (Jiang et al

2019).

The specific mechanism by which P. gingivalis

induce AD has not been elucidated, and the

connection between gingipain caused TREM-1

shredding and tau protein phosphorylation in AD

patients has not been made. Here I proposed a model

to investigate whether P. gingivalis can pass into the

brain from oral infection by assessing the level of

gingipain in the brain of mice. Then, the level of

sTREM-1 in the brain will be determined to confirm

the effect of P. gingivalis on TREM-1 shredding

(Figure 1). To investigate the contribution of

sTREM-1 in the formation of phosphorylated tau

(phospho-tau), Trem-1 deficient mice will be used

and compared with wild type mice for the level of

phospho-tau. Lastly, as Rgp is shown to be

responsible for TREM-1 shredding, the effect of a

Rgp inhibitor will be investigated to prevent mice

infected with P. gingivalis from AD.

626

Zhu, Y.

Gingipain Causes Tau Tangle Formation in Alzheimer’s Disease Brains via Regulation of TREM-1.

DOI: 10.5220/0011250000003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 626-631

ISBN: 978-989-758-595-1

Figure 1: Proposed model for the development of Alzheimer’s disease induced by P. gingivalis (Created with

BioRender.com). P. gingivalis present at periodontal infections enters the brain via an unknown route, but several possible

mechanisms have been proposed (Dominy et al 2019). Once inside the brain, gingipains are secreted and cleaves the TREM1

receptor present on myeloid cells into sTREM1, which then circulates in CSF and amplifies inflammatory responses. sTREM1

also cause rise in tau concentration and increased phosphorylation, which results in neuronal loss that contribute to AD

development.

2 MATERIALS AND METHODS

2.1 Growth of P. Gingivalis

P. gingivalis (W83, ATCC® BAA308™) will be

grown on tryptic soy agar plates under anaerobic

condition in an anerobic gas chamber at 37°C for 3

days (ATCC® Medium 2722). P. gingivalis will then

be harvested from the agar by scrapping and

suspended in PBS + 2% methylcellulose at a

concentration of 1 × 10

cells/ml after washing with

phosphate-buffered saline (PBS). Cell concentration

will be determined by using a counting chamber

under a microscope.

2.2 Animal

Female 5XFAD mice (#34840) from the Jackson

Laboratory will be purchased and used for infection

with P. gingivalis. Mice needs to be maintained under

specific pathogen free conditions and fed with

LabDiet® 5K52 formulation (6% fat) with tap water.

The control and bacterially infected mice will be

maintained in separate cages and kept under a regular

12h dark/light cycle with a temperature and humidity

of 22°C and 60, respectively.

2.3 Generation of Trem-1 Knockout

Mice

The generation of Trem-1 knockout mice will follow

the procedure outlined by Weber et al, 2014 (Weber

et al 2014). Briefly, a vector based on KS loxP ftr Neo

BS cloning vector need to be constructed in order to

delete exon 2 of the Trem-1 gene. Deletion blocks

TREM-1 activity as exon2 encodes the extracellular

region and the ligand-binding site. Additional

restriction sites (AseI and AvaI), and the positive

selection markers, PuroR and Neomycin, is added for

the selection. A Tk counterselection cassette is also

included. The vector needs to be electroporated into

mice embryonic stem cells using a BAC plasmid.

Further mating of the genetically modified mice with

mice carrying Cre recombinase is required to obtain

Trem-1 knockout mice.

Gingipain Causes Tau Tangle Formation in Alzheimer’s Disease Brains via Regulation of TREM-1

627

2.4 P. gingivalis Oral Infection on Mice

Forty-three weeks old 5XFAD female mice were

used for oral infection with P. gingivalis. The mice

will be anaesthetized with ketamine (100mg/kg) and

xylazine (10mg/kg) by intraperitoneal injection. The

eyes will be lubricated with ophthalmic ointment to

prevent drying. Anaesthetized mice will then be tied

around the upper maxillary left and right second

molar with silk ligature (FS 5080, Dolphin Sutures).

100ml of the bacterial solution will be applied to the

buccal surface of the maxillae of the mice in infection

groups. This procedure needs to be repeated every

other day for six weeks. 100ml of PBS + 2%

methylcellulose will be applied to the control groups

on the same days.

Figure 2.

2.5 Effect of P. gingivalis Infection on

the Level of Gingipain in Brain

To study the level of gingipain in the brain of infected

mice, 20 infected and 20 controls of 43 weeks old

female 5XFAD mice were used. Mice brains need to

be prepared as outlined by Clark et al, 2011 (Clark et

al 2011). Briefly, brains extracted were sliced using a

cryostat into 40µm thick sections and fixed onto

slides. Then, immunohistochemistry will be

performed following the procedure described by

Dominy et al, 2019, where the Rgp antibody 18E6

(University of Georgia) needs to be added to the

samples and the result will be visualized using

UltraView Universal DAB Detection System

(Ventana Medical Systems) (Dominy et al 2019).

2.6 Effect of P. gingivalis Infection on

the Level of TREM-1 in Brain

Immune cells will be isolated from the brain of 20

infected and 20 control mice to determine TREM-1

levels. The cells will undergo flow cytometry using

the procedure stated by Liu et al, 2019 (Liu et al

2019). Briefly, cells extracted will be suspended in

Hank’s balanced salt solution (ThermoFisher), and

TREM-1 antibodies (R&D, clone 174031) will be

added. Flowjo (Tree Star Inc.) will then be used to

analyze the results.

2.7 Effect of P. gingivalis Infection on

the Level of Strem-1 in Brain

To investigate the level of sTREM-1 in mice brains,

cerebrospinal fluid (CSF) from 20 infected and 20

control 43 weeks old female 5XFAD mice were used.

CSF will be extracted into polypropylene tubes using

the method proposed by Sakic, 2019 (Šakić 2019).

Then, the samples were analyzed using the TREM1

mouse ELISA kit (EMTREM1, ThermoFisher)

following the protocol.

2.8 Effect of TREM-1 Knockout on the

Level of Total/Phospho-Tau

The level of tau was investigated in 20 infected

TREM-1(+/+) and 20 infected TREM-1(-/-) 5XFAD

mice. Mice will be anaesthetized by intraperitoneal

injection with 4% chloral hydrate (10 mL/kg), and the

level of total tau protein present in the brain of the

mice can be determined using the Tau (total) mouse

ELISA kit (KMB7011, ThermoFisher) following the

protocol using brain homogenate. The level of

phospho-tau protein present can be determined using

the Tau (Phospho) [pS199] Mouse ELISA kit

(KMB7041, ThermoFisher) following the protocol

using brain homogenate. Sample obtained were

analyzed using a spectrophotometer, and the

absorbance is read at 450nm to determine the

concentration.

2.9 Effect of Gingipain Inhibitor on the

Level of Strem-1 and Phospho-Tau

To test the effect of gingipain inhibitor in treating

AD, 40 43 weeks old female 5XFAD mice were

infected with P. gingivalis. Twenty of them received

Rgp inhibitor (A18522, Adooq Bioscience) in DMSO

every day starting from week two by intravenous

injection (10mg/kg). Control mice received only

DMSO. Determine the level of sTREM-1 and

phospho-tau in the brain of the mice as above.

2.10 Data Analysis

All statistical analysis of the data obtained will be

performed using RStudio version 1.2.5042. One-way

analysis of variance (ANOVA) will be performed to

assess whether the difference between the sample

means of the groups were significant. A Bonferroni

test will then be performed to reduce false-positive

results. Data will be considered significant at P<0.05.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

628

3 CONCLUSION

Figure 2: Experimental setup for sample collection of different groups (Created with BioRender.com). A) Negative control

group, receiving only the solvent used to suspend the P. gingivalis cells. Act as a reference for the level of gingipain, TREM1

and tau protein in brain. B) Positive control, mice are infected with P. gingivalis to confirm that the infection will lead to an

increase in TREM1 and tau proteins in brain. C) Mice with TREM1 knockout, to investigate whether TREM1 influences the

level of tau protein in the brain. D) Mice treated with gingipain inhibitor, to investigate whether the effect is due to the

presence of gingipain or not and also act as a possible treatment method for the infection.

This set of experiments could propose a new

mechanism by which AD can be induced. Suppose

the result meets the hypothesis: the bacterially

infected mice have a higher level of gingipain,

sTREM-1 and phosphorylated tau in the brain than

control. In that case, it can be concluded that orally

infected P. gingivalis is able to migrate into the brain

and secretes gingipain that cleaves TREM-1 from cell

surfaces. The increase in the level of sTREM-1

triggers the accumulation and phosphorylation of tau,

which is a sign of AlD. Suppose the inhibitor-treated

mice demonstrated a lower level of sTREM-1 and

phosphor-tau in the brain than positive control mice.

It could be concluded that Rgp is responsible for the

disease, and inhibition of this virulence factor could

prevent AD development. However, other adverse

effects of the replication bacteria inside the brain

have not been determined and may contribute to the

neurodegeneration seen in AD patients, thus

requiring further characterization. This study only

investigates the effect of Rgp, and the role of Kgp in

the pathogenesis of the bacteria would need to be

determined to understand the complete mechanism

by which P. gingivalis causes AD.

Although this set of experiments did not

investigate the route by which P. gingivalis passes

into the brain, but many models have been proposed

and the mechanism of entry may contribute to the

development of AD symptoms. For example, P.

gingivalis may gain entry into the brain via direct

damage to the endothelial cells of the blood-brain

barrier (BBB) via its gingipains. As an earlier study

has demonstrated the ability of gingipains secreted by

the bacteria to induce apoptosis in endothelial cells

(Sheets, Potempa, Travis, Casiano, Fletcher 2005).

The damage to BBB are often seen as an early marker

for the development of AD, and contributes to

neurodegeneration that led to dementia. Thus, both

the effect of the damage of BBB and cleavage of

TREM1 need to be considered to fully characterize P.

gingivalis infection on causing AD.

Overall, the experiments provided a pathway to

investigate a novel cause of AD and presented a

possible treatment method.

Gingipain Causes Tau Tangle Formation in Alzheimer’s Disease Brains via Regulation of TREM-1

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Figure 3: Steps of making brain sections for immunohistochemistry.

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