Oral Treponema denticola infection induces Aβ1-40 and Aβ1-42 accumulation in the hippocampus of C57BL/6 mice

Accumulation of amyloid-β (Aβ) in the brain is a central component of pathology in Alzheimer’s disease. A growing number of evidences demonstrate close associations between periodontal pathogens including Porphyromonas gingivalis (P. gingivalis) and Treponema denticola (T. denticola) and AD. However, the effect and mechanisms of T. denticola on accumulation of Aβ remain to be unclear. In this study, we demonstrated that T. denticola was able to enter brain and act directly on nerve cells resulting in intra and extracellular Aβ 1−40 and Aβ 1−42 accumulation in the hippocampus of C57BL/6 mice by selectively activating both β-secretase and γ-secretase. Furthermore, both KMI1303, an inhibitor of β-secretase, as well as DAPT, an inhibitor of γ- secretase were found to be able to inhibit the effect of T. denticola on Aβ accumulation in N2a neuronal cells. Overall, it is concluded that T. denticola increases the expression of Aβ 1−42 and Aβ 1−40 by its regulation on beta-site amyloid precursor protein cleaving enzyme-1 and Presenilin 1. reactions performed with the Applied Biosystems QuantStudio 6 Flex Real-Time PCR System in the presence of 0.8 μL for each 2 μL of cDNA, 6μL Nuclease-Free water, 0.4 μL ROX Reference Dye II and 10 μL of TB Green Primix Ex Taq all


Introduction
Alzheimer's disease (AD), the most common form of dementia, is characterized by a cerebral accumulation and aggregation of amyloid-β (Aβ) peptides and tau hyperphosphorylation, the main components of plaques and tangles respectively (Scheltens et al. 2016). Aβ oligomers induce AD-like lesions, such as tau phosphorylation or synaptic loss, and accumulate in brain regions related to memory and cognitive function, resulting in related dysfunction (Viola et al. 2015) and recent evidences indicate a role for Aβ as an antimicrobial peptide (Gosztyla et al. 2018). Aβ 1−42 and Aβ 1−40 are the most common toxic subtypes in human body (Wirths et al.2019), which are believed to play a key role in neuronal loss and cognitive dysfunction in AD (Lesné et al. 2006). Aβ is a series of short peptides of 38-43 amino acids, produced by degradation of amyloid precursor protein (APP), which is highly expressed in the central nervous system. APP is rst hydrolyzed into β -N-terminal fragment and β -C-terminal fragment under the action of β -secretase 1 (BACE1), and subsequently hydrolyzed by γ -secretase of which catalytic active center is Presenilin. Presenilin 1 (PS1) and Presenilin 2 (PS2) are two subtypes of presenilin with similar biological functions. The abnormal expression of BACE1, PS1 and PS2 can affect the cleavage of APP, thus affecting the production of Aβ.
Chronic periodontitis (CP) has been identi ed as a signi cant risk factor for the development of AD (Kamer et al.2015). The probability of cognitive impairment in the elderly with alveolar bone resorption is 2.4 times higher than that of those without alveolar bone resorption (Shin et al. 2016). It has been found that the degree of cognitive impairment in patients with severe periodontitis is three times higher than that of patients with mild periodontitis or without periodontitis (Gil-Montoya et al. 2017). The more alveolar bone absorption in the elderly with normal cognitive function, the greater the amount of Aβ deposition in brain tissue. Periodontal pathogens such as P. gingivalis or T. denticola could cause chronic periodontitis and possibly contribute to the clinical onset of AD (Sochocka et al. 2017), with evidence revealing that oral P. gingivalis infection in mice led to brain colonization and increased production of amyloid plaque component Aβ 1− 42 (Dominy et al. 2019). T. denticola, one of the important pathogenic bacteria of CP with a detection rate of 56.8% in CP patients (Sparks et al. 2012) and a member of spirochetes, was detected signi cantly more frequently in brain samples of AD patients (14 of 16), when compared with that of healthy controls (4 of 18) (Riviere et.al 2002, Poole et al. 2013. Thus, we speculate that T. denticola can promote Aβ deposition and pathological process of AD similar to P. gingivalis. However, there is a lack of research about the effect and underlying mechanisms of T. denticola in AD. To verify the aforementioned assumption, oral T. denticola infection was induced in C57BL/6 mice and examined the effect of T. denticola on Aβ in those mice with P. gingivalis as positive control and PBS as negative control. Our study demonstrated that oral T. denticola infection induces Aβ 1−42 and Aβ 1−40 accumulation in the hippocampus of C57BL/6 mice by up regulating BACE1 and PS1. Here, we also reported that both KMI1303 and N-[N-(3,5-Di uorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) could inhibit the effect of T. denticola on Aβ in N2a neuronal cells.

Materials And Methods
2.1. Bacterial strains and growth P. gingivalis ATCC33277 cultures were routinely grown in Brain Heart Infusion (BHI) medium containing 37 g/L Brain Heart Infusion (Becton, Dickinson and Company), L-cysteine hydrochloride (0.5 mg/mL), and hemin (5 μg/mL) under anaerobic conditions;T. denticola ATCC 35405 was grown under anaerobic conditions in new oral spirochete (NOS) broth, as described previously (Fenno et al. 2005). All growth media were incubated in anaerobic conditions for at least 18 h prior to use.

Mice and treatments
The male mice used in this study (The Animal experiment center of SichuanUniversity, Chengdu, China) were of a C57BL/6 background, and at 8 weeks of age. All the mice were divided into three groups randomly, including the experimental group, the positive control group and the blank control group. The experimental group and the positive control group were separately treated with a mixture of 25U 2%CMC and 25U T. denticola or P. gingivalis uid for 24 weeks at a frequency of three times a week (Chukkapalli et al. 2014, Ilievski et al. 2018,while the blank control group received an equal volume of phosphate buffer saline (PBS) three times a week. One week after the nal treatment , mice were anaesthetized. After the collection of blood, mice were quickly perfused intracardiacally with chilled PBS (0.1 M, pH 7.3),then hippocampi, trigeminal ganglionweres and aortas were removed, and the hippocampi were dissected. All trigeminal ganglionweres, aortas and some of the hippocampi were ash frozen in liquid nitrogen and stored at -80℃, the other half of hippocampi were xed with 4%paraformaldehyde. Relative guidelines were followed in our animal experiment.

Polymerase Chain Reaction
To con rm the spread of periodontal pathogens from the mouth to the brain of mice, genomic DNA was isolated from the tissues samples with the DNeasy Blood & Tissue Kit (Qiagen, Germany). DNA ampli cation was performed using a PCR ampli cation kit (TAKARA, Japan) according to the manufacturer's instructions. Brie y, the PCR mixture contained 12.5-μL Taq PCR Master, 0.5-μL (10 μg/mL) DNA samples, 1-μL forward primer, 1-μL reverse primer, and 10-μL sterilized ddH2O. The primers used for ampli cation were as follows: P. gingivalis, 5′-AGGCAGCTTGCCATACTGCG-3′ (forward) 5′-ACTGTTAGCAACTACCGATGT-3′ (reverse) and T. denticola, 5′-TAATACCGAATGTGCTCATTTACAT-3′ (forward) 5′-CTGCCATATCTCTATGTCATTGCTCTT-3′(reverse) (TSINGKE, China). The sequencing parameters were an initial denaturation step at 94•C for 4 min and 35 cycles involving (94 • C for 30 s), annealing (55 • C for 5 s), and elongation(72 • C for 10 s) . The PCR products were analyzed using agarose gel electrophoresis (1.5%) under 100 V for 23 min with P. gingivalis ATCC 33277 DNA and T. denticola ATCC 35405 DNA as the positive control and a blank reaction system as the negative control. The speci c bands of the samples were compared with the positive group to determine whether the samples contained P. gingivalis ATCC 33277 and T. denticola ATCC 35405.

Immunohistochemistry
The tissues of the mice hippocampi xed by 4% paraformaldehyde were embedded in para n and sliced coronally into 5-μm sections using a vibratome (Leica, Germany) . The sections were performed for antigen repair at 99.9 ° C for 30 min after depara nation and then incubated for 10 min in 3% H2O2, and incubated with primary rabbit polyclonal antibodies against Aβ 1-40 (bs-0877R, 1:100, Boiss, China), primary Rabbit monoclonal antibodies against Aβ 1-42 (ab224275, 1:200, Abcam, Cambridge, MA, USA) overnight at 4 °C. Then the sections were incubated with biotinlabeled secondary antibodies (1:300, 865002, R&D Systems) for 30 min at 37 °C and the Cell and Tissue Staining Kit (CTS005, Anti-Rabbit HRP-DAB System, R&D Systems) was used to detect the positive staining area. The images were captured by a camera (Nikon, 90i, Tokyo, Japan). And 3 sections per mouse were being counted in a blind manner.

Cell culture and Treatment
N2a neuronal cells were cultured in Dulbecco's Modi ed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1 U/Ml penicillin, and 1 m g/mL streptomycin in a humidi ed (5% CO 2 , 37 ℃) incubator. After con rming the cell status, the N2a cells were rst seeded in 24-well tissue culture plates at a density of 10 5 cells per well in DMEM culture medium with 10% fetal bovine serum. After cells adhesion, N2a cells were treated with 1 μmol/L DAPT ( ApexBio Technology, USA) which was used as γsecretase inhibitor or treated with 1 μmol/L KMI1303(WaKo, Japan) for 6 h, or cultured with no treatment.
After that, cells were infected with T. denticola or P. gingivalis at a density of 10 7 CFU per well for 2h.

Statistical Analysis
Data were presented as the mean ± standard deviation and analyzed by SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Student's t-test was applied to analyze statistical differences. The level of signi cance was set at p < 0.05 (*).

3.1.T. denticola 16S rDNA was detected in aorta, trigeminal ganglionwere and hippocampus of mice
Since Spirochetes like Treponema pallidium are capable of entering brain tissue and pathologies suggest a relationship with AD (Miklossy et al. 2016). Besides, P. gingivalis was identi ed in the brain of Alzheimer's disease patients (Dominy et al. 2019). Thus, after 24 weeks of oral injection, Polymerase Chain Reaction was employed to detect whether T. denticola and P. gingivalis entered the hippocampus. As presented in Fig.1, T. denticola and P. gingivalis were detected signi cantly more frequently in hippocampus (7 of 10), compared with blank control samples (0 of 10). To detect how T. denticola and P. gingivalis entered the hippocampus, the aortas and trigeminal ganglion were subjected to detection. T. denticola was found in 7 aorta samples, while P. gingivalis was found in 9 samples. Further, 3 trigeminal ganglion samples were revealed to contain T. denticola

T. denticola induced Aβ 1-40 and Aβ 1-42 accumulation in hippocampus
We wondered whether T. denticola or P. gingivalis, which entered the hippocampus, had an effect on Aβ, so we subjected to ELISA analysis and immunohistochemistrym using the PBS group as blank control and the P. gingivalis group as positive control. Our data revealed that oral T. denticola or P. gingivalis infection induced Aβ 1-40 and Aβ 1-42 accumulation in the hippocampus of mice, and there was no signi cant difference between the two groups ( Fig.2b and c). Overexpressed Aβ 1-40 and Aβ 1-42 mainly accumulated in the cytoplasm and intercellular substance.
3.3.T. denticola induced Aβ 1-40 and Aβ 1-42 accumulation by directly acting on mouse nerve cells The above experiments revealed that T. denticola and P. gingivalis entered the hippocampus and the expression of Aβ 1-40 and Aβ 1-42 increased in the hippocampus of mice. We speculated that T. denticola and P. gingivalis entering the hippocampus directly could act on mouse nerve cells and cause Aβ 1-40 and Aβ 1-42 accumulation. To test our hypothesis, we stimulated N2a neurons with T. denticola and P.
gingivalis in vitro. The immuno uorescence results con rmed our hypothesis. As shown in Figure.3, the expression of Aβ 1-40 and Aβ 1-42 in N2a cells co cultured with T. denticola or P. gingivalis was signi cantly increased compared with the blank control group.
3.4. T. denticola induced Aβ accumulation by up regulating BACE1 and PS1 , KMI-1303 and DAPT inhibited the effect of T.denticola on Aβ For the purpose of further exploring the mechanism of T. denticola on Aβ, total RNA extracted from the brain tissues was used for real-time PCR. It was shown that the expressions of BACE1, PS1 and PS2 increased in T. denticola and P. gingivalis groups. The expression level of BACE1 and PS1 in T. denticola group was signi cantly higher than that in the blank control group. Next, we subjected to Western blot analysis. The results of Western blot showed that the expression levels of BACE1 and PS1 in T. denticola and P. gingivalis groups were signi cantly higher than those in blank control group. In addition to the effect on β -secretase and PS1, P. gingivalis had an effect on PS2, a subtype of presenilin with similar biological functions to PS1. In order to further con rm the mechanism of T. denticola inducing Aβ accumulation, β-secretase inhibitor and γ-secretase inhibitor were used to test and verify the results above in vitro. KMI-1303, the inhibitor with an IC50 value of 9 nM, has the same effect and mechanisms as KMI429 (Asai et al. 2006) on inhibiting β-secretase activity accoding to the manufacturer. PS1 and PS2 are two subtypes of presenilin and have similar biological functions, and the effect of T. denticola on PS1 was more signi cant. Thus we choose DAPT , a potent γ-secretase inhibitor which can lead to a decreased exibility of key PS1 regions related to the recognition and internalization of γ-secretase substrates (Aguayo-Ortiz et al. 2019) to con rm the mechanisms of T. denticola on Aβ. As presented in Fig. 4 the expression of Aβ 1-42 in the inhibitor group is signi cantly lower than that in the coculture group.

Discussion
CP is known to trigger several human diseases including AD (Kamer et al.2015), Atherosclerosis (Arabi et al. 2018) etc. The periodontal pathogens such as P. gingivalis and T. denticola have been found in human post-mortem brain tissues of Alzheimer's Disease patients (Riviere et.al 2002, Poole et al. 2013 (Tang et al. 2020) and neuroin ammation (Tang et al. 2020). The viewpoint that spirochetes are involved in the possible etiology of AD was rst proposed by Miklossy (Miklossy et al. 1993). Moreover, in a more recent compended analysis of brain and blood samples from 495 samples from AD patients, 91% were positive for spirochetes compared to 0% for 185 controls (Miklossy et al. 2011). However, the effect and mechanisms of T. denticola, a periodontal pathogen as well as a spirochete in AD was not clearly understood. Here, we used P. gingivalis oral infected mouse model (Dominy et al. 2019 ) as a positive control to study the effect of T. denticola. Our experiment demonstrated that P. gingivalis and T. denticola could enter the brain mainly through the blood and cause Aβ aggregation to the same extent . Besides, T. denticola 16S rDNA was also detected in a small number of trigeminal ganglion samples. Hence we speculate T. denticola may directly enter the hippocampus via blood and trigeminal nerves to induce Aβ 1-40 and Aβ 1-42 accumulation.
Cerebral accumulation and aggregation of Aβ peptides is a characteristic pathological marker of AD patients. Soluble Aβ oligomers are believed to represent key structures that produce cytotoxicity, contribute to synaptic de cits and initiate the detrimental cascade involved in the pathology of AD (Salahuddin et al. 2016, Larson et al. 2012, Klein et al. 2006. BACE1 cleavage of APP is the rate-limiting step along the amyloidogenic processing pathway (O'Brien et al. 2011) and PS1 is involved in γ-secretase activity and in uences Aβ 1-40 and Aβ 1-42 production. Knockout of BACE1 completely blocks the generation of Aβ (Cai et al. 2001). About 90% of mutations in human PS1 by individually lead to reduced production of Aβ 1-40 and Aβ 1-42 (Sun et al. 2017). Our data indicate that T. denticola increases Aβ 1-40 and Aβ 1-42 accumulation in the hippocampus of C57BL/6 mice by upregulating BACE1 and PS1. Besides, we have veri ed the above results with β-secretase inhibitor and γ-secretase inhibitor in N2a neuronal cells.
In summary, we present data obtained by PCR, ELISA, IHC, qPCR, Western blot and IF to support the hypothesis that T. denticola in oral cavity can enter the hippocampus via the blood and trigeminal ganglion and act directly on nerve cells resulting in intra and extracellular Aβ 1-40 and Aβ 1-42 accumulation in the hippocampus of C57BL/6 mice. The up-regulation of BACE1 and PS1 is the mechanism of Aβ 1-40 and Aβ 1-42 increase induced by T. denticola is.

Declarations
Data Availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.