Cathepsins and SARS-CoV-2 infection: From pathogenic factors to potential therapeutic targets.

Coronavirus disease-19 (COVID-19) is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. The COVID-19 pandemic began in March 2020 and has wrought havoc on health and economic systems worldwide. Efficacious treatment for COVID-19 is lacking: Only preventive measures as well as symptomatic and supportive care are available. Preclinical and clinical studies have indicated that lysosomal cathepsins might contribute to the pathogenesis and disease outcome of COVID-19. Here, we discuss cutting-edge evidence on the pathological roles of cathepsins in SARS-CoV-2 infection, host immune dysregulations, and the possible underlying mechanisms. Cathepsins are attractive drug targets because of their defined substrate-binding pockets, which can be exploited as binding sites for pharmaceutical enzyme inhibitors. Accordingly, the potential modulatory strategies of cathepsin activity are discussed. These insights could shed light on the development of cathepsin-based interventions for COVID-19.

Cathepsin H: Molecular characteristics and clues to function and mechanism.

Cathepsin H (CatH) is a lysosomal cysteine protease with a unique aminopeptidase activity that is extensively expressed in the lung, pancreas, thymus, kidney, liver, skin, and brain. Owing to its specific enzymatic activity, CatH has critical effects on the regulation of biological behaviours of cancer cells and pathological processes in brain diseases. Moreover, a neutral pH level is optimal for CatH activity, so it is expected to be active in the extra-lysosomal and extracellular space. In the present review, we describe the expression, maturation, and enzymatic properties of CatH, and summarize the available experimental evidence that mechanistically links CatH to various physiological and pathological processes. Finally, we discuss the challenges and potentials of CatH inhibitors in CatH-induced disease therapy.

Emerging Roles of Microglia in Blood-brain Barrier Integrity in Aging and Neurodegeneration.

The blood-brain barrier (BBB) is a highly selective interface between the blood and the brain parenchyma. It plays an essential role in maintaining a specialized environment for central nervous system function and homeostasis. The BBB disrupts with age, which contributes to the development of many age-related disorders due to central and peripheral toxic factors or BBB dysfunction. Microglia, the resident innate immune cells of the brain, have recently been explored for their ability to directly and indirectly regulate the integrity of the BBB. This review will focus on the current understanding of the molecular mechanisms utilized by microglia to regulate BBB integrity and how this becomes disrupted in aging and age-associated diseases. We will also discuss the rational to consider microglia as a therapeutic target to prevent or slow down the neurodegeneration.

Role of PG0192 and PG0193 in the modulation of pro-inflammatory cytokines in macrophages in response to Porphyromonas gingivalis.

Porphyromonas gingivalis is the main pathogen of chronic periodontitis. However, the specific mechanisms through which P. gingivalis induces immune and inflammatory responses in periodontitis have not been completely elucidated. In this study, we investigated the effects of the P. gingivalis outer membrane protein OmpH (encoded by PG0192 and PG0193) on interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression in macrophages to assess the pro-inflammatory cytokine responses. A PG0192-PG0193 deletion mutant strain and a com△PG0192-0193 strain were constructed. Furthermore, rOmpH-1 and rOmpH-2 encoded by PG0192 and PG0193, respectively, were cloned, expressed, and purified for subsequent experiments. Notably, the expression of IL-6 and TNF-α at mRNA and protein levels was downregulated upon treatment of macrophages with the PG0192-PG0193 deletion mutant strain, whereas treatment of macrophages with P. gingivalis W83 co-incubated with rOmpH-1 or rOmpH-2 upregulated IL-6 and TNF-α mRNA levels. The addition of C5aR antagonist blocked this induction. Overall, our findings provided important insights into the roles of PG0192 and PG0193 for promoting IL-6 and TNF-α expression in macrophages exposed to P. gingivalis and revealed the involvement of C5aR in the pro-inflammatory response.

GSK3ß is involved in promoting Alzheimer's disease pathologies following chronic systemic exposure to Porphyromonas gingivalis lipopolysaccharide in amyloid precursor proteinNL-F/NL-F knock-in mice.

In line with the strong association between periodontitis and Alzheimer's disease (AD) clinically, preclinical studies have shown that systemic exposure to Porphyromonas gingivalis (Pg) initiates AD pathologies. However, the involvement of periodontitis in promoting AD pathologies is unclear. In the present study, we provided evidence that chronic systemic exposure to lipopolysaccharide derived from Pg (PgLPS, 1 mg/kg, daily, intraperitoneally) prompted neuroinflammation and tau hyperphosphorylation in 10-month-old of amyloid precursor protein (APP) knock-in mice, a model of AD, carrying the Swedish and Beyreuther/Iberian mutation (APPNL-F/NL-F). The learning and memory function were assessed using the passive avoidance test. The production of APP, Amyloid (A)ß1-42, cytokines, synaptic proteins and the activation of glycogen synthase kinase (GSK)-3ß as well as phosphorylation of tau were analyzed by immunohistochemistry, Western blotting or an enzyme-linked immunosorbent assay (ELISA) in the cortex of APPNL-F/NL-F mice. We found that systemic exposure of PgLPS for three consecutive weeks induced learning and memory deficits with significantly reduced postsynaptic density protein (PSD95). Increased hyperphosphorylation of tau in multiple residues, including Ser202, Thr231 and Ser396, but not the accumulation of Aß1-42 was detected in the neurons of APPNL-F/NL-F mice. Furthermore, PgLPS increased the GSK3ß activity by reducing its phosphorylation of the serine residue at position 9 (Ser9) and promoted neuroinflammation by increasing the expression of interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF-α) while decreasing that of interleukin-10 (IL-10) and transforming growth factor (TGFß) in the cortex of APPNL-F/NL-F mice. Moreover, the PgLPS-increased GSK3ß activity was detected in both microglia and neurons, while the PgLPS-increased TNF-α expression was mainly detected in the microglia in the cortex of APPNL-F/NL-F mice. In in vitro studies, PgLPS (1 µg/ml) stimulation increased the mRNA and protein level of TNF-α in MG6 microglia, which were significantly inhibited by the GSK3ß-specific inhibitor TWS119. In contrast, the tau hyperphosphorylation and activation of GSK3ß in N2a neurons were enhanced after treatment with conditioned medium from PgLPS-stimulated microglia, which was attenuated after pre-treatment with TNF-α inhibitor. Taken together, these findings indicate that GSK3ß is involved in prompting microglia (TNF-α)-dependent tau hyperphosphorylation in neurons, resulting in learning and memory deficits in APPNL-F/NL-F mice without changes in the Aß expression during chronic systemic exposure to PgLPS. We propose that dampening GSK3ß activation may help delay the periodontitis-promoted pathological progression of AD.

Porphyromonas Gingivalis Infection Induces Synaptic Failure via Increased IL-1ß Production in Leptomeningeal Cells.

BACKGROUND: Studies have reported that synaptic failure occurs before the Alzheimer's disease (AD) onset. The systemic Porphyromonas gingivalis (P. gingivalis) infection is involved in memory decline. We previously showed that leptomeningeal cells, covering the brain, activate glial cells by releasing IL-1ß in response to systemic inflammation. OBJECTIVE: In the present study, we focused on the impact of leptomeningeal cells on neurons during systemic P. gingivalis infection. METHODS: The responses of leptomeningeal cells and cortical neurons to systemic P. gingivalis infection were examined in 15-month-old mice. The mechanism of IL-1ß production by P. gingivalis infected leptomeningeal cells was examined, and primary cortical neurons were treated with P. gingivalis infected leptomeningeal cells condition medium (Pg LCM). RESULTS: Systemic P. gingivalis infection increased the expression of IL-1ß in leptomeninges and reduced the synaptophysin (SYP) expression in leptomeninges proximity cortex in mice. Leptomeningeal cells phagocytosed P. gingivalis resulting in lysosomal rupture and cathepsin B (CatB) leakage. Leaked CatB mediated NLRP3 inflammasome activation inducing IL-1ß secretion in leptomeningeal cells. Pg LCM decreased the expression of synaptic molecules, including SYP, which was inhibited by an IL-1 receptor antagonist pre-treatment. CONCLUSION: These observations demonstrate that P. gingivalis infection is involved in synaptic failure by inducing CatB/NLRP3 inflammasome-mediated IL-1ß production in leptomeningeal cells. The periodontal bacteria-induced synaptic damage may accelerate the onset and cognitive decline of AD.

A novel cyclic peptide (Naturido) modulates glia-neuron interactions in vitro and reverses ageing-related deficits in senescence-accelerated mice.

The use of agents that target both glia and neurons may represent a new strategy for the treatment of ageing disorders. Here, we confirmed the presence of the novel cyclic peptide Naturido that originates from a medicinal fungus (Isaria japonica) grown on domestic silkworm (Bombyx mori). We found that Naturido significantly enhanced astrocyte proliferation and activated the single copy gene encoding the neuropeptide VGF and the neuron-derived NGF gene. The addition of the peptide to the culture medium of primary hippocampal neurons increased dendrite length, dendrite number and axon length. Furthermore, the addition of the peptide to primary microglial cultures shifted CGA-activated microglia towards anti-inflammatory and neuroprotective phenotypes. These findings of in vitro glia-neuron interactions led us to evaluate the effects of oral administration of the peptide on brain function and hair ageing in senescence-accelerated mice (SAMP8). In vivo analyses revealed that spatial learning ability and hair quality were improved in Naturido-treated mice compared with untreated mice, to the same level observed in the normal ageing control (SAMR1). These data suggest that Naturido may be a promising glia-neuron modulator for the treatment of not only senescence, but also Alzheimer's disease and other neurodegenerative diseases.

Systemic Exposure to Lipopolysaccharide from Porphyromonas gingivalis Induces Bone Loss-Correlated Alzheimer's Disease-Like Pathologies in Middle-Aged Mice.

BACKGROUND: Alzheimer's disease (AD) and bone loss are clinically exacerbated. However, the mechanism of exacerbation remains understood. OBJECTIVE: We tested our hypothesis that periodontitis is involved in the exacerbation, contributing to AD pathologies. METHODS: The bone, memory, and inflammation in bone and brain were examined in 12-month-old mice after systemic exposure to lipopolysaccharide from Porphyromonas gingivalis (P gLPS) for 3 consecutive weeks. RESULTS: Compared with control mice, bone loss in tibia (26% decrease) and memory decline (47% decrease) were induced in mice with a positive correlation after exposure to P gLPS (r = 0.7378, p = 0.0011). The IL-6 and IL-17 expression in tibia was negatively correlated with the bone volume/total tissue volume (r = -0.6619, p = 0.0052; r = -0.7129, p = 0.0019), while that in the cortex was negatively correlated with the memory test latency (r = -0.7198, p = 0.0017; p = 0.0351, r = -0.5291). Furthermore, the IL-17 expression in microglia was positively correlated with Aß42 accumulation in neurons (r = 0.8635, p < 0.0001). In cultured MG6 microglia, the P gLPS-increased IL-6 expression was inhibited by a PI3K-specific inhibitor (68% decrease), and that of IL-17 was inhibited by IL-6 antibody (41% decrease). In cultured N2a neurons, conditioned medium from P gLPS-stimulated microglia (MCM) but not P gLPS increased the productions of AßPP, CatB, and Aß42, which were significantly inhibited by pre-treatment with IL-17 antibody (67%, 51%, and 41% decrease). CONCLUSION: These findings demonstrated that chronic systemic exposure to P gLPS simultaneously induces inflammation-dependent bone loss and AD-like pathologies by elevating IL-6 and IL-17 from middle age, suggesting that periodontal bacteria induce exacerbation of bone loss and memory decline, resulting in AD progression.

Cathepsin B inhibition blocks neurite outgrowth in cultured neurons by regulating lysosomal trafficking and remodeling.

Lysosomes are known to mediate neurite outgrowth in neurons. However, the principal lysosomal molecule controlling that outgrowth is unclear. We studied primary mouse neurons in vitro and found that they naturally develop neurite outgrowths over time and as they did so the lysosomal cysteine protease cathepsin B (CTSB) mRNA levels dramatically increased. Surprisingly, we found that treating those neurons with CA-074Me, which inhibits CTSB, prevented neurites. As that compound also inhibits another protease, we evaluated a N2a neuronal cell line in which the CTSB gene was deleted (CTSB knockout, KO) using CRISPR technology and induced their neurite outgrowth by treatment with retinoic acid. We found that CTSB KO N2a cells failed to produce neurite outgrowths but the wild-type (WT) did. CA-074Me is a cell permeable prodrug of CA-074, which is cell impermeable and a specific CTSB inhibitor. Neurite outgrowth was and was not suppressed in WT N2a cells treated with CA-074Me and CA-074, respectively. Lysosome-associated membrane glycoprotein 2-positive lysosomes traffic to the plasma cell membrane in WT but not in CTSB KO N2 a cells. Interestingly, no obvious differences between WT and CTSB KO N2a cells were found in neurite outgrowth regulatory proteins, PI3K/AKT, ERK/MAPK, cJUN, and CREB. These findings show that intracellular CTSB controls neurite outgrowth and that it does so through regulation of lysosomal trafficking and remodeling in neurons. This adds valuable information regarding the physiological function of CTSB in neural development.

The Critical Role of IL-10 in the Antineuroinflammatory and Antioxidative Effects of Rheum tanguticum on Activated Microglia.

Rheum tanguticum Maxim. ex Balf. (Rt), a traditional Tibetan medicine, is known to exert various bioactivities, including anti-inflammatory and antioxidative activities. The present study was conducted to investigate anti-inflammatory and antioxidative effects of Rt on activated microglia. Rt (10 µg/ml) significantly inhibited the mean protein level of interleukin-1ß (IL-1ß) in the organotypic hippocampal slice cultures following treatment with chromogranin A (CGA, 10 nM) and pancreastatin (10 nM), endogenous microglial activators present in senile plaques. Rt also significantly inhibited the expression and production of inflammatory and oxidative molecules, including IL-1ß, tumor necrosis factor-α, and nitric oxide, by cultured microglia after treatment with CGA. These effects of Rt are considered to be mediated by the secretion of interleukin-10 (IL-10) from microglia, because neutralizing antibodies against IL-10 significantly canceled these effects. To explore the causative components of Rt responsible for inducing the secretion of IL-10, the effects of seven components of Rt on the IL-10 expression in microglia were examined. Among them, aloe-emodin (10 µM) and (+)-catechin (30 µM) were able to induce the secretion of IL-10 from cultured microglia. Therefore, aloe-emodin and (+)-catechin are deemed responsible for the antineuroinflammatory and antioxidative effects of Rt through the secretion of IL-10 from microglia. Accordingly, Rt is considered potentially useful for the treatment of AD.