P. gingivalis-LPS Induces Mitochondrial Dysfunction Mediated by Neuroinflammation through Oxidative Stress

Porphyromonas gingivalis (P. gingivalis), a key pathogen in periodontitis, is associated with neuroinflammation. Periodontal disease increases with age; 70.1% of adults 65 years and older have periodontal problems. However, the P. gingivalis- lipopolysaccharide (LPS)induced mitochondrial dysfunction in neurodegenerative diseases remains elusive. In this study, we investigated the possible role of P. gingivalis-LPS in mitochondrial dysfunction during neurodegeneration. We found that P. gingivalis-LPS treatment activated toll-like receptor (TLR) 4 signaling and upregulated the expression of Alzheimer’s disease-related dementia and neuroinflammatory markers. Furthermore, the LPS treatment significantly exacerbated the production of reactive oxygen species and reduced the mitochondrial membrane potential. Our study highlighted the pivotal role of P. gingivalis-LPS in the repression of serum response factor (SRF) and its co-factor p49/STRAP that regulate the actin cytoskeleton. The LPS treatment repressed the genes involved in mitochondrial function and biogenesis. P. gingivalis-LPS negatively altered oxidative phosphorylation and glycolysis and reduced total adenosine triphosphate (ATP) production. Additionally, it specifically altered the mitochondrial functions in complexes I, II, and IV of the mitochondrial electron transport chain. Thus, it is conceivable that P. gingivalis-LPS causes mitochondrial dysfunction through oxidative stress and inflammatory events in neurodegenerative diseases.


Introduction
Periodontitis is one of the most common oral chronic inflammatory diseases that is triggered by bacterial microorganisms. Increasing evidence indicates the correlation between chronic periodontitis and dementia [1,2]. One of the known periodontal bacterial species enriched in periodontitis disease is Porphyromonas gingivalis (P. gingivalis), an oral gram-negative anaerobe. It is one of the keystone species in the development of periodontal disease and an important factor responsible for various systemic diseases associated with aging, mainly neurodegenerative diseases, by promoting the development of Aβ plaques, cognitive impairment, perturbed motor control, and dementia [3]. P. gingivalis has the potential to induce neuroinflammation via its intracerebral entry or entry of its virulence factors through various direct and indirect penetration mechanisms [4,5]. It has a wide variety of virulence factors, including lipopolysaccharide (LPS), lipoteichoic acids, outer membrane vesicles (OMVs), gingipains and fimbriae [6]. LPS, a bacterial endotoxin, is a major constituent of the outer membrane of P. gingivalis and reaches the neuronal cells through the OMVs, where it interacts with pattern recognition receptors, such as toll-like receptors (TLR) 2 and 4 [7]. It plays a critical role in mediating inflammation and stimulating cells to secrete pro-inflammatory cytokines, mainly IL-1β, IL-6, TNF-α, NO, and reactive oxygen species (ROS) through toll-like receptor (TLR) 4 and nuclear factor-κB [8][9][10]. It is well documented that LPS derived from different bacterial species, e.g., P. gingivalis-LPS The overproduction of nitric oxide by intracellular NO synthase was observed after the treatment ( Figure 1B). The relative mRNA expression of Alzheimer's disease-related dementia (ADRD) biomarkers, T-Tau (Total-Tau), VEGF, and TGF-β was significantly increased ( Figure 1C). The neuroinflammatory markers iNOS, IL-1β, IL-6, and TNF-α were also detected at high levels ( Figure 1D). Thus, P. gingivalis-LPS induced pathogenesis of AD and ADRD markers through neuroinflammation.

P. gingivalis-LPS Induces Mitochondrial ROS and Decreases Membrane Potential Mediated by TLR4
P. gingivalis-LPS treatment specifically activated the TLR4 mRNA expression, and its expression was recovered using the TLR4 inhibitor CLI-095 (Supplementary Figure S2). The ROS production was elevated significantly with 10.0 µg/mL LPS treatment. The treatment of CLI-095, along with the LPS, prevented ROS production ( Figure 2A). With the increased ROS production, the membrane potential was significantly decreased, as indicated by the shift from red to green fluorescence with the LPS treatment ( Figure 2B). CLI-095, along with the LPS, retrieved the membrane potential as well. Constitutively, 4-HNE expression was increased during the LPS treatment ( Figure 2C). expression was recovered using the TLR4 inhibitor CLI-095 (Supplementary Figure S2). The ROS production was elevated significantly with 10.0 μg/mL LPS treatment. The treatment of CLI-095, along with the LPS, prevented ROS production ( Figure 2A). With the increased ROS production, the membrane potential was significantly decreased, as indicated by the shift from red to green fluorescence with the LPS treatment ( Figure 2B). CLI-095, along with the LPS, retrieved the membrane potential as well. Constitutively, 4-HNE expression was increased during the LPS treatment ( Figure 2C).

P. gingivalis-LPS Downregulates Serum Response Factor and p49/STRAP
P. gingivalis-LPS affects the actin cytoskeleton, and SRF regulates the actin [28,29]. We sought to investigate the effect of P. gingivalis-LPS treatment on SRF and co-factor p49/STRAP. The mRNA and protein expression levels of SRF and p49/STRAP were repressed with 10.0 µg/mL LPS ( Figure 3A-C). Thus, P. gingivalis-LPS downregulated the expression levels of both SRF and p49/STRAP.

P. gingivalis-LPS Downregulates Serum Response Factor and p49/STRAP
P. gingivalis-LPS affects the actin cytoskeleton, and SRF regulates the actin [28,29]. We sought to investigate the effect of P. gingivalis-LPS treatment on SRF and co-factor p49/STRAP. The mRNA and protein expression levels of SRF and p49/STRAP were repressed with 10.0 μg/mL LPS ( Figure 3A-C). Thus, P. gingivalis-LPS downregulated the expression levels of both SRF and p49/STRAP.

P. gingivalis-LPS Alters Oxidative Phosphorylation, Glycolysis and Reduces the ATP
The LPS treatment significantly altered the parameters of mitochondrial function, as evidenced by increased oxygen consumption rate (OCR), which resulted in increased basal respiration, maximal respiratory capacity, and spare respiratory capacity ( Figure 5A). In the glycolytic pathway, basal glycolysis was not affected, but the compensatory glycolysis was increased with the treatment ( Figure 5B). In addition, the ATP production from

P. gingivalis-LPS Alters Oxidative Phosphorylation, Glycolysis and Reduces the ATP
The LPS treatment significantly altered the parameters of mitochondrial function, as evidenced by increased oxygen consumption rate (OCR), which resulted in increased basal respiration, maximal respiratory capacity, and spare respiratory capacity ( Figure 5A). In the glycolytic pathway, basal glycolysis was not affected, but the compensatory glycolysis was increased with the treatment ( Figure 5B). In addition, the ATP production from OCR and glycolysis (ECAR) was reduced with the LPS treatment ( Figure 5C). CLI-095, along with P. gingivalis-LPS treatment, significantly improved OCR, ECAR, and ATP production, which demonstrated TLR4-dependent LPS function.

P. gingivalis-LPS Specifically Alters the Mitochondrial Function in Complex I, II, and IV
High-resolution respiratory analysis using Oxygraph O2k in the intact SH-SY5Y cells treated with LPS confirms the dysfunction of mitochondrial OCR (Supplementary Figure S3). With the substrate-inhibitor titration, we specifically targeted the different complexes of the Electron transport chain (ETC). The LPS-treated cells had increased respiration rates in complex I, II and IV, but complex III was unaffected ( Figure 6).

P. gingivalis-LPS Specifically Alters the Mitochondrial Function in Complex I, II, and IV
High-resolution respiratory analysis using Oxygraph O2k in the intact SH-SY5Y cells treated with LPS confirms the dysfunction of mitochondrial OCR (Supplementary Figure  S3). With the substrate-inhibitor titration, we specifically targeted the different complexes of the Electron transport chain (ETC). The LPS-treated cells had increased respiration rates in complex I, II and IV, but complex III was unaffected ( Figure 6).

TLR4 Expression, Actin Assembly and Mitochondrial Morphology
LPS treatment activated the TLR4 expression ( Figure 7B) compared to the untreated cells ( Figure 7A). In f-actin phalloidin staining, the actin filaments were altered with 10.0 μg/mL LPS treated cells ( Figure 7D), whereas the actin filaments were arranged intact in the untreated cells ( Figure 7C). Furthermore, P. gingivalis-LPS treatment reduces the mitochondrial mass in the MitoTracker staining ( Figure 7F) as compared to untreated control cells ( Figure 7E).

TLR4 Expression, Actin Assembly and Mitochondrial Morphology
LPS treatment activated the TLR4 expression ( Figure 7B) compared to the untreated cells ( Figure 7A). In f-actin phalloidin staining, the actin filaments were altered with 10.0 µg/mL LPS treated cells ( Figure 7D), whereas the actin filaments were arranged intact in the untreated cells ( Figure 7C). Furthermore, P. gingivalis-LPS treatment reduces the mitochondrial mass in the MitoTracker staining ( Figure 7F) as compared to untreated control cells ( Figure 7E).

Discussion
Recent research has revealed the important correlation between mitochondrial dysfunction in the pathophysiology of neurodegenerative diseases [38,39]. The role of mitochondria during neuroinflammation and neurodegeneration has unraveled mitochondria-related immunometabolic processes that may serve as promising therapeutic targets for AD and ADRD [40,41]. P. gingivalis-LPS can induce neuroinflammation and lead to the progression of neuropathological changes. However, the definite mechanisms of P. gingivalis-LPS-mediated mitochondrial dysfunction remain under-explored. Our study utilized undifferentiated SH-SY5Y cells to explore the hypothesis that LPS-mediated mitochondrial dysfunction could be the origin of oxidative stress in neurodegenerative diseases.

Discussion
Recent research has revealed the important correlation between mitochondrial dysfunction in the pathophysiology of neurodegenerative diseases [38,39]. The role of mitochondria during neuroinflammation and neurodegeneration has unraveled mitochondriarelated immunometabolic processes that may serve as promising therapeutic targets for AD and ADRD [40,41]. P. gingivalis-LPS can induce neuroinflammation and lead to the progression of neuropathological changes. However, the definite mechanisms of P. gingi-valis-LPS-mediated mitochondrial dysfunction remain under-explored. Our study utilized undifferentiated SH-SY5Y cells to explore the hypothesis that LPS-mediated mitochondrial dysfunction could be the origin of oxidative stress in neurodegenerative diseases.
P. gingivalis-mediated inflammasome activity and neuroinflammation have been shown to be activated in AD brains [42,43]. Herein, we demonstrated the increased expression of soluble Aβ 1-42 peptide, T-Tau protein, VEGF and TGF-β after P. gingivalis-LPS treatment. It also upregulated the expression of several neuroinflammatory markers such as intracellular NOS, iNOS, IL-1β, IL-6 and TNF-α (Figure 1). These observations validate the potential role of P. gingivalis-LPS in amyloidogenesis, tauopathy and in neuroinflammation. Next, we tested whether TLR2 or TLR4 or both were activated with P. gingivalis-LPS treatment. Our findings indicated that P. gingivalis-LPS acted exclusively through TLR4. (Supplementary Figure S2 and Figure 7A,B). Others have also shown that LPS-induced neuroinflammation is mediated by the activation of the TLR4 signaling pathway [44,45]. In several studies, it has been reported that neurons can express both TLR2 and TLR4, indicating a critical role of these receptors in neuroinflammatory responses [46,47]. Oxidative stress induces neuroinflammation and neurodegeneration [48]. The identification of P. gingivalis-LPS-induced ROS accumulation, reduced MMP, and elevated protein expression of 4-HNE in neuroblastoma cells underscores an important finding ( Figure 2). Moreover, it has been reported that LPS from P. gingivalis increases oxidative stress in periodontal ligament fibroblasts [49] as well as in brain endothelial cells [50].
P. gingivalis and its LPS regulate cellular cytoskeleton dynamics in different cell types [28,29]. We sought to investigate the effects of P. gingivalis-LPS on the transcriptional activity of SRF and its co-factor p49/STRAP. SRF is a dispensable transcription factor for cellular growth, maintaining the cellular cytoskeleton, and it mediates mitochondrial function [30,31]. Interestingly, we found that P. gingivalis-LPS repressed the expression of both SRF and co-factor p49/STRAP (Figure 3), suggesting its role in altered actin morphology and mitochondrial dynamics ( Figure 7C,D). Others have also shown that SRF regulates the actin cytoskeleton [51].
PGC1-α and PGC1-β are transcriptional coactivators and serve as the main regulators of mitochondrial biogenesis and function [52]. Several transcription factors, including NRF-1 and 2, TFAM, are activated by PGC1-α to increase the transcription of genes related to mitochondrial biogenesis and function [53]. Our study showed that P. gingivalis-LPS downregulated the expression of PGC1-α, PGC1-β, NRF-1 and TFAM, and mitochondrial fission and fusion genes and repressed the gene expression of complex-I genes ( Figure 4). The downregulation of PGC1-α has been reported in various inflammatory conditions [53] and negatively regulated the genes involved in mitochondrial biogenesis and function [54].
Next, we investigated the functional aspects of mitochondrial oxidative phosphorylation and glycolysis. The OCR was significantly increased with P. gingivalis-LPS treatment ( Figure 5A). It has been reported that P. gingivalis-LPS treatment significantly increased respiration rates because of oxidative stress in human gingival fibroblast cells [55]. This study demonstrated that with LPS treatment, H 2 O 2 production was enhanced, and ATP generation was reduced, which has been linked to increased oxygen demand through increased mitochondrial respiration (55). In glycolytic parameters, basal glycolysis was unaffected, while compensatory glycolysis was increased ( Figure 5B). Furthermore, total ATP production (glyco-ATP and mito-ATP) was greatly reduced with LPS treatment ( Figure 5C). Others have also reported reduced ATP production in both human gingival fibroblast and endothelial cells after P. gingivalis and its LPS treatment [55,56]. In addition, P. gingivalis-LPS significantly altered the mitochondrial respiration in complex-I, II, and IV but did not affect complex-III of the electron transport chain ( Figure 6). Complex-I, II, and III have been reported as major producers of significant amounts of ROS [23][24][25], but our findings suggest that complex-I and II might be responsible for producing ROS and thereby inducing oxidative stress and mitochondrial dysfunction. It has been demonstrated that mitochondrial dysfunction has been linked to the increased oxygen demand due to increased mitochondrial respiration, which tilts the balance towards preferential ROS production instead of ATP [55] and hence worsens neuroinflammation.
In conclusion, our study has provided evidence that P. gingivalis-LPS triggered oxidative stress resulting in mitochondrial dysfunction and neuroinflammation in SH-SY5Y cells. Interestingly, TLR4-specific inhibitor CLI-095 recovered the LPS-induced mitochondrial dysfunction (Figures 2 and 5). CLI-095 has been tested clinically for antimetastatic effects [57]. Our results suggest that CLI-095 could potentially be useful as a therapeutic agent against P. gingivalis-LPS-mediated neuroinflammation, oxidative stress and mitochondrial dysfunction in AD and ADRD. However, future studies are required to completely understand the mechanisms by which P. gingivalis-LPS would reach the cytosol of the stimulated cells to produce injury and whether CLI-095 could inhibit its adverse effects in-vivo.

Cell Culture, Cell Viability and LDH Assay
The SH-SY5Y cell line and all the cell culture reagents were previously described [58]. The ultrapure P. gingivalis-LPS and CLI-095 were obtained from InvivoGen, San Diego, CA, USA, and used as per the manufacturer's instructions. Cell viability towards P. gingivalis-LPS was determined by using an MTS assay as described [54]. CytoTox 96 ® cytotoxicity assay was used to quantify the lactate dehydrogenase released in the cell culture supernatant after 24 h of P. gingivalis-LPS treatment (CytoTox 96 ® , Promega, Madison, WI, USA). The absorbance at 490 nm was measured with the microplate reader (BioTek Synergy H1).

Quantification of Aβ 1-42 , NOS and Reverse-Transcriptase qPCR
The quantitative analysis of Aβ 1-42 was determined by using a human amyloid beta (aa1-42) quantikine ELISA kit, as per the manufacturer's instructions (R&D Systems). Intracellular NOS activity was determined using a commercially available kit per the manufacturer's instructions (Intracellular NOS Assay Kit, Abcam, Cambridge, UK, ab211085). The fluorescence intensity was determined at Ex/Em = 485/530 nm (BioTek Synergy H1). RT-qPCR was performed as previously described [59]. The primer sequences used to quantify the gene expression are described in Supplementary Table S1.

Flow Cytometry
The mtROS levels were quantified by MitoSOX Red (ThermoFisher, Eugene, OR, USA; M36008). Briefly, SH-SY5Y cells were seeded at a density of 1 × 10 6 cells/well in a 6-well plate and treated with 10.0 µg/mL concentration of P. gingivalis-LPS for 24 h. CLI-095 (1 µM) was added to the cells 1 h before treatment, used alone and in combination with P. gingivalis-LPS treatment [60]. After 24 h, cells were stained with 5 µM MitoSOX Red for 10 min. JC-1 fluorescent dye (5 µg/mL), an indicator of mitochondrial membrane potential (MMP), was used to stain the cells for 10 min (ThermoFisher, Eugene, OR, USA; T3168). The cells were centrifuged and washed gently three times with warm HBSS buffer, followed immediately by flow cytometry analysis. The fluorescence intensity was quantified by a flow cytometer (BD LSRFortessa TM , Franklin Lakes, NJ, USA) and analyzed by FlowJo_v10.8.1 software.

Mitochondrial Oxygen Consumption Rate, Glycolysis and ATP Production
SH-SY5Y cells were seeded at a density of 5 × 10 4 cells/well in XFe96 Well plates (Seahorse Bioscience, Billerica, MA, USA). The cells were treated with a 10.0 µg/mL concentration of P. gingivalis-LPS for 24 h. After treatment, cells were subjected to extracellular flux analysis using the XF Cell Mito Stress Test Kit (Agilent, Santa Clara, CA, USA; 103015-100), XF Glycolytic Rate Assay Kit (Agilent, Santa Clara, CA, USA; 103344-100), and XF Real-Time ATP Rate Assay (Agilent, Santa Clara, CA, USA; 103592-100) respectively.

High-Resolution Respirometry
The Oxygraph 2k (O2K) respirometer (Oroboros Instruments GmbH, Innsbruck, Austria) was utilized to examine the mitochondrial function in the intact cells (2.5 × 10 6 /sample), as previously described [54,61]. Furthermore, the OCR in the complexes was measured according to the substrate-inhibitor-titration protocol as described [54]. Briefly, 5 × 10 6 cells were incubated with digitonin (Sigma, Milwaukee, WI, USA; D5628; 8 µM/million cells) and prepared in MiRO5 buffer for 20 min at 4 • C to permeabilize the cells. Data analysis was performed with DatLab 6.2 software (Innsbruck, Austria), and the OCR of intact cells and from each individual mitochondrial complex was expressed as oxygen flux (pmol/s*Million Cells).

Quantification and Statistical Analysis
Statistical analysis was carried out using GraphPad Prism 9.1.1 Software Inc. (Dotmatics, Boston, MA, USA). Data represent mean values ± SD of at least 3 independent experiments, or as indicated by n. The 2-tailed Student's t-test was used to determine the statistical significance between 2 samples originating from the same population, and the statistical significance between multiple groups was determined by 1-way ANOVA followed by Tukey's multiple comparison test (* p < 0.05; ** p <0.01; *** p < 0.001, **** p < 0.0001, ns: non-significant).

Data Availability Statement:
The raw data are available without reservation upon reasonable request.