A mixture of extracts from natural ingredients reduces the neurotoxic polarization of microglia via modulating NF-κB/NF-E2-related factor 2 activation.

Neurotoxic microglia-provoked neuroinflammation is implicated in cognitive decline in Alzheimer's disease (AD). Supplementation with Ginkgo biloba, phosphatidylserine, Curcuma longa, and propolis is reported to improve the cognitive functions of elderly people; however, the underlying mechanisms of this combination of natural ingredients are unknown. We investigated the effects of a mixture of extracts from propolis, Coffea arabica, Gotu kola, phosphatidylserine, Ginkgo biloba, and Curcuma longa (mixture) on microglia polarization after exposure to amyloid ß1-42 (Aß1-42, 1 µM) and lipopolysaccharide from Porphyromonas gingivalis (PgLPS, 1 µg/mL), using MG6 and BV2 microglial cells. Exposure to Aß1-42 and PgLPS (AL) raised the mRNA expression of IL-1ß, TNF-α, and IL-6, nuclear translocation of p65 NF-κB in MG6 cells and BV2 cells, and mitochondrial reactive oxygen species (ROS) production in MG6 cells. The mixture dramatically suppressed the mRNA expression of IL-1ß, TNF-α, and IL-6, but significantly promoted that of IL-10, TGFß1, and BDNF in AL-exposed MG6 and BV2 cells. Furthermore, the mixture significantly suppressed the nuclear translocation of p65 NF-κB but significantly promoted that of NF-E2-related factor 2 (Nrf2) in AL-exposed MG6 and BV2 cells. Furthermore, the mixture significantly ameliorated mitochondrial ROS production but increased mitochondrial membrane potential in MG6 cells. These observations strongly suggest that the mixture demotes the neuropathic polarization of microglia by modulating NF-κB/Nrf2 activation and improving mitochondrial functions. This study supplies the potential mechanisms of the efficacy of a combination of natural ingredients that can be applied in the prevention of cognitive decline in AD and aging by targeting microglia-mediated neuroinflammation.

211At on gold nanoparticles for targeted radionuclide therapy application.

Targeted alpha therapy (TAT) is a methodology that is being developed as a promising cancer treatment using the α-particle decay of radionuclides. This technique involves the use of heavy radioactive elements being placed near the cancer target area to cause maximum damage to the cancer cells while minimizing the damage to healthy cells. Using gold nanoparticles (AuNPs) as carriers, a more effective therapy methodology may be realized. AuNPs can be good candidates for transporting these radionuclides to the vicinity of the cancer cells since they can be labeled not just with the radionuclides, but also a host of other proteins and ligands to target these cells and serve as additional treatment options. Research has shown that astatine and iodine are capable of adsorbing onto the surface of gold, creating a covalent bond that is quite stable for use in experiments. However, there are still many challenges that lie ahead in this area, whether they be theoretical, experimental, and even in real-life applications. This review will cover some of the major developments, as well as the current state of technology, and the problems that need to be tackled as this research topic moves along to maturity. The hope is that with more workers joining the field, we can make a positive impact on society, in addition to bringing improvement and more knowledge to science.

Gingipain-carrying outer membrane vesicles from Porphyromonas gingivalis cause barrier dysfunction of Caco-2 cells by releasing gingipain into the cytosol.

Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.

Human ß-Defensin 3 Inhibition of P. gingivalis LPS-Induced IL-1ß Production by BV-2 Microglia through Suppression of Cathepsins B and L.

Cathepsin B (CatB) is thought to be essential for the induction of Porphyromonas gingivalis lipopolysaccharide (Pg LPS)-induced Alzheimer's disease-like pathologies in mice, including interleukin-1ß (IL-1ß) production and cognitive decline. However, little is known about the role of CatB in Pg virulence factor-induced IL-1ß production by microglia. We first subjected IL-1ß-luciferase reporter BV-2 microglia to inhibitors of Toll-like receptors (TLRs), IκB kinase, and the NLRP3 inflammasome following stimulation with Pg LPS and outer membrane vesicles (OMVs). To clarify the involvement of CatB, we used several known CatB inhibitors, including CA-074Me, ZRLR, and human ß-defensin 3 (hBD3). IL-1ß production in BV-2 microglia induced by Pg LPS and OMVs was significantly inhibited by the TLR2 inhibitor C29 and the IκB kinase inhibitor wedelolactonne, but not by the NLRPs inhibitor MCC950. Both hBD3 and CA-074Me significantly inhibited Pg LPS-induced IL-1ß production in BV-2 microglia. Although CA-074Me also suppressed OMV-induced IL-1ß production, hBD3 did not inhibit it. Furthermore, both hBD3 and CA-074Me significantly blocked Pg LPS-induced nuclear NF-κB p65 translocation and IκBα degradation. In contrast, hBD3 and CA-074Me did not block OMV-induced nuclear NF-κB p65 translocation or IκBα degradation. Furthermore, neither ZRLR, a specific CatB inhibitor, nor shRNA-mediated knockdown of CatB expression had any effect on Pg virulence factor-induced IL-1ß production. Interestingly, phagocytosis of OMVs by BV-2 microglia induced IL-1ß production. Finally, the structural models generated by AlphaFold indicated that hBD3 can bind to the substrate-binding pocket of CatB, and possibly CatL as well. These results suggest that Pg LPS induces CatB/CatL-dependent synthesis and processing of pro-IL-1ß without activation of the NLRP3 inflammasome. In contrast, OMVs promote the synthesis and processing of pro-IL-1ß through CatB/CatL-independent phagocytic mechanisms. Thus, hBD3 can improve the IL-1ß-associated vicious inflammatory cycle induced by microglia through inhibition of CatB/CatL.

The Role of Cysteine Protease Cathepsins B, H, C, and X/Z in Neurodegenerative Diseases and Cancer.

Papain-like cysteine proteases are composed of 11 human cysteine cathepsins, originally located in the lysosomes. They exhibit broad specificity and act as endopeptidases and/or exopeptidases. Among them, only cathepsins B, H, C, and X/Z exhibit exopeptidase activity. Recently, cysteine cathepsins have been found to be present outside the lysosomes and often participate in various pathological processes. Hence, they have been considered key signalling molecules. Their potentially hazardous proteolytic activities are tightly regulated. This review aims to discuss recent advances in understanding the structural aspects of these four cathepsins, mechanisms of their zymogen activation, regulation of their activities, and functional aspects of these enzymes in neurodegeneration and cancer. Neurodegenerative effects have been evaluated, particularly in Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and neuropsychiatric disorders. Cysteine cathepsins also participate in tumour progression and metastasis through the overexpression and secretion of proteases, which trigger extracellular matrix degradation. To our knowledge, this is the first review to provide an in-depth analysis regarding the roles of cysteine cathepsins B, H, C, and X in neurodegenerative diseases and cancer. Further advances in understanding the functions of cysteine cathepsins in these conditions will result in the development of novel, targeted therapeutic strategies.

Zinc finger protein 335 mediates lipopolysaccharide-induced neurodegeneration and memory loss as a transcriptional factor in microglia.

Zinc finger protein 335 (Zfp335) is a transcription factor that regulates mammalian neurogenesis and neuronal differentiation. It is a causative factor for severe microcephaly, small somatic size, and neonatal death. Here, we evaluated the effects of Zfp335 in the adult mouse brain after lipopolysaccharide (LPS) challenge. We used wild-type (WT) and Zfp335 knock-down (Zfp335+/- ) mice with LPS administered in the intracerebral ventricle in vivo and cultured microglia treated with LPS in vitro. The impact of Zfp335 was evaluated by RT-PCR, RNA-sequencing, western blotting, immunocytochemistry, ELISA, and the memory behavior tests. Knockdown of Zfp335 expression ameliorated microglia activation significantly, including reduced mRNA and protein expression of Iba1, reduced numbers of microglia, reduced cell diameter, and increased branch length, in the brains of 2-month-old mice after LPS treatment. Zfp335 was expressed in microglia and neurons, but increased in microglia, not neurons, in the brain of mice after LPS administration. LPS-induced microglia-mediated neurodegeneration was dependent upon microglial Zfp335 controlled by nuclear factor-kappa B. Microglial Zfp335 affected neuronal activity through transcriptional regulation of lymphocyte antigen-6M (Ly6M). Our data suggest that Zfp335 is a key transcription factor that exacerbates microglia-mediated neurodegeneration through upregulation of Ly6M expression. Inhibition of microglial Zfp335 may be a new strategy for preventing brain disease induced by microglia activation.

Association between human papillomavirus particle production and the severity of recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) has a wide range of severity. We investigate the relationship between human papillomavirus (HPV) particle production and severity of RRP. From September 2005 to June 2021, 68 RRP samples (from 29 patients) were included. HPV type was determined. HPV viral load, physical status, and demographic and clinical characteristics were assessed. Immunohistochemistry (IHC) was performed for p16, Ki-67, L1, and E4. We used NanoSuit-CLEM (correlative light and electron microscopy) and transmission electron microscopy (TEM) to examine the samples. The total number of surgeries in HPV-positive and HPV-negative cases were 3.78 (n = 55/68, range: 1-16) and 1.30 (n = 13/68, range: 1-3), respectively (p = 0.02). IHC showed that L1 and E4 were correlated and expressed on the tumour surface. NanoSuit-CLEM and TEM revealed HPV particles in L1-positive nuclei. L1 IHC-positive cases had a shorter surgical interval (p < 0.01) and more frequent surgeries (p = 0.04). P16 IHC, viral load, and physical status were not associated with disease severity. This study visualised HPV particle production in RRP for the first time. Persistent HPV particle infection was associated with severity. We suggest L1 IHC for evaluating RRP severity in addition to the Derkay score.

Targeting cellular gaps using Janus nanoparticles containing cationic polymers and surfactant lipids.

Since nanoparticles are taken up into cells by endocytosis, phagocytosis, or pinocytosis, they have been studied as intracellular drug carriers. Janus particles have an anisotropic structure composed of two or more distinct domains and have been proposed for use in various applications, including use as imaging agents or nanosensors. This study aimed to clarify the influence of the type of nanoparticles on their distribution in a human Caucasian colon adenocarcinoma (Caco-2) cell monolayer. We fabricated Janus and conventional spherical nanoparticles composed of pharmaceutically applicable ingredients. Janus and spherical nanoparticles composed of a cationic polymer and surfactant lipids were prepared by controlling the solvent removal pattern from the oil phase in the solvent removal process using the solvent evaporation and solvent diffusion methods. The distribution of nanoparticles in the Caco-2 cell monolayer was then evaluated using confocal laser microscopy. The mean hydrodynamic size of the fabricated Janus nanoparticles was 119.2 ± 4.6 nm. Distribution analysis using Caco-2 cells suggested that Janus nanoparticles were localized around the adherens junctions located just below the tight junction. Clear localization was not observed in non-Janus nanoparticles with the same composition. The clear localization of the Janus nanoparticles around the adherens junction may be due to their positive charge and asymmetric structure. Our results suggest the considerable potential for the development of nanoparticulate drug carriers to target cellular gaps.

The Dual Nature of Microglia in Alzheimer's Disease: A Microglia-Neuron Crosstalk Perspective.

Microglia are critical players in the neuroimmune system, and their involvement in Alzheimer's disease (AD) pathogenesis is increasingly being recognized. However, whether microglia play a positive or negative role in AD remains largely controversial and the precise molecular targets for intervention are not well defined. This partly results from the opposing roles of microglia in AD pathology, and is mainly reflected in the microglia-neuron interaction. Microglia can prune synapses resulting in excessive synapse loss and neuronal dysfunction, but they can also promote synapse formation, enhancing neural network plasticity. Neuroimmune crosstalk accelerates microglial activation, which induces neuron death and enhances the microglial phagocytosis of ß-amyloid to protect neurons. Moreover, microglia have dual opposing roles in developing the major pathological features in AD, such as amyloid deposition and blood-brain barrier permeability. This review summarizes the dual opposing role of microglia in AD from the perspective of the interaction between neurons and microglia. Additionally, current AD treatments targeting microglia and the advantages and disadvantages of developing microglia-targeted therapeutic strategies are discussed.

Human ß-Defensin 3 Inhibits Porphyromonas Gingivalis Lipopolysaccharide-Induced Oxidative and Inflammatory Responses of Microglia by Suppression of Cathepsins B and L.

Recently, the effects of antibacterial peptides are suggested to have therapeutic potential in Alzheimer's disease. Furthermore, systemic treatment of Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) induced Alzheimer's disease-like neuropathological changes in middle-aged mice. Then, we examined whether human ß-defensins (hBDs), antimicrobial peptides produced by the oral mucosa and salivary glands, can suppress Pg LPS-induced oxidative and inflammatory responses by microglia. hBD3 (1 µM) significantly suppressed Pg LPS-induced production of nitric oxide and interleukin-6 (IL-6) by MG6 cells, a mouse microglial cell line. hBD3 (1 µM) also significantly inhibited Pg LPS-induced expression of IL-6 by HMC3 cells, a human microglial cell line. In contrast, neither hBD1, hBD2 nor hBD4 failed to inhibit their productions. Furthermore, hBD3 suppressed Pg LPS-induced p65 nuclear translocation through the IκBα degradation. Pg LPS-induced expression of IL-6 was significantly suppressed by E64d, a cysteine protease inhibitor, and CA-074Me, a known specific inhibitor for cathepsin B, but not by pepstatin A, an aspartic protease inhibitor. Interestingly, hBD3 significantly inhibited enzymatic activities of recombinant human cathepsins B and L, lysosomal cysteine proteases, and their intracellular activities in MG6 cells. Therefore, hBD3 suppressed oxidative and inflammatory responses of microglia through the inhibition of cathepsins B and L, which enzymatic activities are necessary for the NF-κB activation.

Update on Findings about Sudden Sensorineural Hearing Loss and Insight into Its Pathogenesis.

Sudden sensorineural hearing loss (SSNHL) is routinely encountered and is one of the most common emergent diseases in otolaryngology clinics. However, the etiology of SSNHL remains unclear. Due to the inaccessibility of the living human inner ear for biopsy, studies investigating the etiology of SSNHL have been performed by analyzing data obtained from examinations using peripheral blood or imaging. We updated the findings obtained from serological, magnetic resonance imaging, genetic, and viral examinations to reveal the etiology of SSNHL. Regarding viral examination, we focused on sensorineural hearing loss associated with coronavirus disease (COVID-19) because the number of correlated reports has been increasing after the outbreak. The updated findings revealed the following three possible mechanisms underlying the development of SSNHL: thrombosis and resulting vascular obstruction in the cochlea, asymptomatic viral infection and resulting damage to the cochlea, and cochlear inflammation and resulting damage to the cochlea. Thrombosis and viral infection are predominant, and cochlear inflammation can be secondarily induced through viral infection or even thrombosis. The findings about sensorineural hearing loss associated with COVID-19 supported the possibility that asymptomatic viral infection is one of the etiologies of SSNHL, and the virus can infect inner ear tissues and directly damage them.

ATP13A2 modifies mitochondrial localization of overexpressed TOM20 to autolysosomal pathway.

Mutations in ATP13A2 cause Kufor-Rakeb Syndrome (KRS), a juvenile form of Parkinson's Disease (PD). The gene product belongs to a diverse family of ion pumps and mediates polyamine influx from lysosomal lumen. While the biochemical and structural studies highlight its unique mechanics, how PD pathology is linked to ATP13A2 function remains unclear. Here we report that localization of overexpressed TOM20, a mitochondrial outer-membrane protein, is significantly altered upon ATP13A2 expression to partially merge with lysosome. Using Halo-fused version of ATP13A2, ATP13A2 was identified in lysosome and autophagosome. Upon ATP13A2 co-expression, overexpressed TOM20 was found not only in mitochondria but also within ATP13A2-containing autolysosome. This modification of TOM20 localization was inhibited by adding 1-methyl-4-phenylpyridinium (MPP+) and not accompanied with mitophagy induction. We suggest that ATP13A2 may participate in the control of overexpressed proteins targeted to mitochondrial outer-membrane.

Direct identification of the charge state in a single platinum nanoparticle on titanium oxide.

A goal in the characterization of supported metal catalysts is to achieve particle-by-particle analysis of the charge state strongly correlated with the catalytic activity. Here, we demonstrate the direct identification of the charge state of individual platinum nanoparticles (NPs) supported on titanium dioxide using ultrahigh sensitivity and precision electron holography. Sophisticated phase-shift analysis for the part of the NPs protruding into the vacuum visualized slight potential changes around individual platinum NPs. The analysis revealed the number (only one to six electrons) and sense (positive or negative) of the charge per platinum NP. The underlying mechanism of platinum charging is explained by the work function differences between platinum and titanium dioxide (depending on the orientation relationship and lattice distortion) and by first-principles calculations in terms of the charge transfer processes.

Auditory and Vestibular Characteristics of NLRP3 Inflammasome Related Autoinflammatory Disorders: Monogenic Hearing Loss Can Be Improved by Anti-interleukin-1 Therapy.

Inflammasomes are large multimeric protein complexes which regulate the activation of the proinflammatory cytokines interleukins-1ß and-18 and inflammatory cell death called pyroptosis. NLRP1, NLRP3, NLRC4, AIM2, and pyrin can induce the formation of inflammasomes. Of these, the NLRP3 inflammasome is the most well-characterized. Recent studies revealed that variants of the NLRP3 gene cause genetic diseases, including systemic inflammatory syndrome called cryopyrin-associated periodic syndrome (CAPS) and non-syndromic sensorineural hearing loss DFNA34. NLRP3 variants cause CAPS and DFNA34 by constitutively activating the NLRP3 inflammasome and increasing IL-1ß release. Patients with CAPS show systemic inflammatory symptoms, and hearing loss is a characteristic feature. Patients with CAPS and DFNA34 show progressive bilateral sensorineural hearing loss. Hearing loss has unique characteristics that can be improved or stabilized by anti-interluekin-1 therapy, although it is usually difficult to alleviate genetic hearing loss by drugs. However, it should be noted that there is a window of opportunity to respond to treatment, and younger patients are most likely to respond. It is important to know the characteristics of CAPS and DFNA34 for early diagnosis, and mutation analysis of NLRP3 will lead to a definite diagnosis. In this review, we summarize the current understanding of the mechanisms of the NLRP3 inflammasome and characteristics of patients with CAPS and DFNA34, especially focused on auditory and vestibular findings.

Therapeutic Devices for Motor Symptoms in Parkinson's Disease: Current Progress and a Systematic Review of Recent Randomized Controlled Trials.

Background: Pharmacotherapy is the first-line treatment option for Parkinson's disease, and levodopa is considered the most effective drug for managing motor symptoms. However, side effects such as motor fluctuation and dyskinesia have been associated with levodopa treatment. For these conditions, alternative therapies, including invasive and non-invasive medical devices, may be helpful. This review sheds light on current progress in the development of devices to alleviate motor symptoms in Parkinson's disease. Methods: We first conducted a narrative literature review to obtain an overview of current invasive and non-invasive medical devices and thereafter performed a systematic review of recent randomized controlled trials (RCTs) of these devices. Results: Our review revealed different characteristics of each device and their effectiveness for motor symptoms. Although invasive medical devices are usually highly effective, surgical procedures can be burdensome for patients and have serious side effects. In contrast, non-pharmacological/non-surgical devices have fewer complications. RCTs of non-invasive devices, especially non-invasive brain stimulation and mechanical peripheral stimulation devices, have proven effectiveness on motor symptoms. Nearly no non-invasive devices have yet received Food and Drug Administration certification or a CE mark. Conclusion: Invasive and non-invasive medical devices have unique characteristics, and several RCTs have been conducted for each device. Invasive devices are more effective, while non-invasive devices are less effective and have lower hurdles and risks. It is important to understand the characteristics of each device and capitalize on these.

Extralysosomal cathepsin B in central nervous system: Mechanisms and therapeutic implications.

Cathepsin B (CatB) is a typical cysteine lysosomal protease involved in a variety of physiologic and pathological processes. It is expressed in most cell types and is primarily localized within subcellular endosomal and lysosomal compartments. Emerging scientific evidence indicates that lysosomal leaked CatB is involved in mitochondrial stress, inflammasome activation, and nuclear senescence, but without the acidic environment. CatB is also secreted as a myokine, which is involved in muscle-brain cross talk and neuronal dendritic remodeling. Lysosomal-leaked and cellular-secreted CatB functions are dependent on its enzymatic activity at a neutral pH. In the present review, we summarize the available experimental evidence that mechanistically links extralysosomal CatB to physiological and pathological functions in central nervous system, and their potential for use in therapeutic approaches.

Microglial cathepsin E plays a role in neuroinflammation and amyloid ß production in Alzheimer's disease.

Regulation of neuroinflammation and ß-amyloid (Aß) production are critical factors in the pathogenesis of Alzheimer's disease (AD). Cathepsin E (CatE), an aspartic protease, is widely studied as an inducer of growth arrest and apoptosis in several types of cancer cells. However, the function of CatE in AD is unknown. In this study, we demonstrated that the ablation of CatE in human amyloid precursor protein knock-in mice, called APPNL-G-F mice, significantly reduced Aß accumulation, neuroinflammation, and cognitive impairments. Mechanistically, microglial CatE is involved in the secretion of soluble TNF-related apoptosis-inducing ligand, which plays an important role in microglia-mediated NF-κB-dependent neuroinflammation and neuronal Aß production by beta-site APP cleaving enzyme 1. Furthermore, cannula-delivered CatE inhibitors improved memory function and reduced Aß accumulation and neuroinflammation in AD mice. Our findings reveal that CatE as a modulator of microglial activation and neurodegeneration in AD and suggest CatE as a therapeutic target for AD by targeting neuroinflammation and Aß pathology.

Novel prognostic value and potential utility of opioid receptor gene methylation in liquid biopsy for oral cavity cancer.

Opioids are a class of recreational drugs and prescription medications that bind to a group of G-protein-coupled receptors known as opioid receptors (ORs). ORs are involved in the development of many types of cancer; however, their role in head and neck squamous cell carcinoma (HNSCC) is complex and poorly understood. Here, we analyzed the methylation status of five OR genes in verification (301 HNSCC primary samples) and validation (five circulating tumor DNA [ctDNA] samples) studies using quantitative methylation-specific PCR (Q-MSP). OPRL1 and OPRM1 methylation levels were significantly higher in HNSCC tissues than in corresponding normal tissues from the same individuals (P = 0.001 and P < 0.001, respectively). In Kaplan-Meier estimate and multivariate Cox proportional hazard analyses, two genes (OPRL1 and OPRM1) were significantly associated with increased recurrence in the methylation group with oral cavity cancer. Furthermore, a validation study of ctDNA demonstrated that OPRL1 genes exhibited predictive performance as emerging biomarkers and were each capable of discriminating the plasma from tumor-free individuals. We characterized the relationship between OR gene methylation status and outcomes in oral cavity cancer. Our results highlight the potential utility of ctDNA methylation-based detection in the clinical management of oral cavity cancer.

Secreted gingipains from Porphyromonas gingivalis increase permeability in human cerebral microvascular endothelial cells through intracellular degradation of tight junction proteins.

Despite a clear correlation between the infiltration of periodontal pathogens in the brain and cognitive decline in Alzheimer's disease (AD), the precise mechanism underlying bacteria crossing the blood-brain barrier (BBB) remains unclear. The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteases termed gingipains. Gingipains appear to be key virulence factors that exacerbate sporadic AD. We herein report that gingipains are involved in increasing permeability of hCMEC/D3 cell monolayer, human cerebral microvascular endothelial cell lines, through degradation of tight junction proteins including Zonula occludens-1 (ZO-1) and occludin. There was a significant decrease in the mean protein levels of ZO-1 and occludin after infection of hCMEC/D3 cells with wild-type (WT) P. gingivalis. However, infection of these cells with a gingipain-deficient P. gingivalis strain showed significantly lower reduction of the mean protein levels of either ZO-1 and occludin, compared to the WT strain. Similar results were obtained after treatment with culture supernatant from WT and gingipain-deficient P. gingivalis strains. In vitro digestion of human recombinant ZO-1 and occludin by WT P. gingivalis culture supernatant in the absence or presence of gingipain inhibitors indicated that gingipains directly degraded these tight junction proteins. A close immunohistochemical examination using anti-gingipain antibody further revealed that gingipains localized in the cytosol and nuclei of hCMEC/D3 cells after infection with WT P. gingivalis and treatment with its culture supernatant. Furthermore, intracellular localization of outer membrane vesicles (OMVs) bound gingipains from WT P. gingivalis and OMV-induced degradation of ZO-1 and occludin were also observed in hCMEC/D3 cells. Thus, the delivery of gingipains into the cerebral microvascular endothelial cells, probably through OMV, may be responsible for the BBB damage through intracellular degradation of ZO-1 and occludin.