Mast cells are heterogeneous tissue-resident immune cells, with connective tissue mast cells (CTMCs) and mucosal mast cells in rodents exhibiting distinct phenotypes and activation profiles. Cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily adhesion molecule, has been implicated in mast cell-nerve and mast cell-stromal interactions as well as in the pathogenesis of atopic dermatitis. Here, we investigated CADM1 function in CTMCs using a monoclonal antibody against the extracellular domain of CADM1, termed 3E1. CTMCs were differentiated from bone marrow-derived mast cells (BMMCs) by fibroblast coculture, where CADM1 expression was markedly upregulated compared with BMMCs. Treatment with 3E1 downregulated CADM1 expression and suppressed β-hexosaminidase release from IgE-sensitized, antigen-stimulated CTMCs by 18%, whereas BMMCs were unaffected. In addition, 3E1 reduced FM4-64-positive granule formation in activated CTMCs by 34.2% and 27.3% at 5 and 60 min, respectively. Confocal analysis further showed that 3E1 pretreatment decreased F-actin rearrangement in activated CTMCs by 66.2% at 5 min. In a passive cutaneous anaphylaxis model, intravenous 3E1 reduced dermal mast cell degranulation by 10.4%. These findings identify CADM1 as a regulator of IgE-mediated degranulation in CTMCs and demonstrate 3E1 as a valuable tool for dissecting mast cell heterogeneity in relation to tissue localization.

BACKGROUND: Stroke-prone spontaneously hypertensive rats (SHRSP) exhibit slow-twitch muscle-specific hypotrophy compared with normotensive Wistar-Kyoto rats (WKY). Because slow-twitch muscles are prone to disuse atrophy, SHRSP may experience both disuse atrophy and impaired recovery from it. This study investigated the response of SHRSP to disuse atrophy and subsequent recovery, using WKY as a control. RESULTS: WKY and SHRSP were subjected to a 7-day tail suspension followed by reloading for 1, 3, and 7 days. The soleus of WKY and SHRSP showed similar atrophic rates following tail suspension; however, the recovery after reloading was delayed in SHRSP. Moreover, WKY, but not SHRSP, exhibited sarcomere structure disruption after tail suspension, followed by necrosis, inflammatory cell infiltration, and edema upon reloading. Phosphorylation of ribosomal protein S6 (RPS6), an indicator of protein translation, was significantly higher in tail-suspended WKY-but not SHRSP-than those in non-tail-suspended groups after reloading. p70-S6 kinase 1 (S6K1), an upstream protein of RPS6, was phosphorylated at Thr389 in a mechanistic target of rapamycin complex 1-dependent manner to the same extent in both WKY and SHRSP; however, the expression of p60-S6K1-a shorter isoform of p70-S6K1 that activates RPS6 without p60-S6K1 phosphorylation-significantly increased only in tail-suspended WKY compared with those in non-tail-suspended WKY and tail-suspended SHRSP. Previously, p60-S6K1 protein expression was thought to result from an alternative translation of the full-length S6K1 transcript that also produces other S6K1 isoforms. However, recent studies have identified a p60-S6K1-specific transcript, and our PCR results showed that this p60-S6K1-specific transcript, but not the full-length S6K1 transcript, was significantly increased only in tail-suspended WKY corresponding with the increase of p60-S6K1 protein expression. CONCLUSIONS: SHRSP exhibited different phenotypes in disuse atrophy and recovery from it compared with WKY, which could be related to suppressed RPS6 phosphorylation associated with the lack of upregulation in p60-S6K1 expression. These findings suggest that p60-S6K1, in cooperation with p70-S6K1, activates RPS6 and promotes rapid recovery from disuse atrophy by enhancing the transcription of the p60-S6K1-specific transcript. The study also suggests a potential involvement of hypertension in disuse atrophy and its recovery.

OBJECTIVE: Cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, is abundantly expressed on nerve fibers. Recently, the anti-CADM1 ectodomain antibody 3E1 has been shown to be potentially useful as a local anesthetic due to its high affinity for subcutaneous nerve fibers. When injected intrathecally into mice, where 3E1 accumulates, and whether it serves as an analgesic was examined. METHODS: The 3E1 injection was detected using indocyanine green-based imaging and immunohistochemistry. As a neuropathic pain model, mechanical allodynia-affected mice were created by spinal nerve ligation (SNL). These mice were administered intrathecally with 3E1, control antibody, or pregabalin, and then the pain threshold of the hind paw was measured using von Frey monofilaments. After SNL, followed by intrathecal injection of 3E1, real-time PCR was conducted to examine the expression of pain-related genes, interleukin (IL)-1β, IL-2, IL-6, IL-10, tumor necrosis factor-α, chemokine (C-C motif) ligand (CCL) 2, CCL7, and CCL11. RESULTS: Intrathecally injected 3E1 was localized mainly on the dorsal root ganglion cell bodies and spinal dorsal horn nerve fibers, with no motor side effects. In the von Frey test, 3E1-injected mice exhibited higher pain thresholds for up to 2 weeks compared to control mice. Pregabalin also raised the threshold, but the effect dissipated within 1 day. While SNL increased mRNA expression of all pain-related genes examined in spinal dorsal horn neurons, 3E1 injection suppressed the increased expression of IL-6. CONCLUSION: 3E1 was suggested to be a potential ganglion-blocking analgesic and spinal anesthetic that acts ten or more times longer than pregabalin.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its S1 spike protein to bind to angiotensin-converting enzyme 2 (ACE2) on human cells in the first step of cell entry. Tryptanthrin, extracted from leaves of the indigo plant, Polygonum tinctorium, using d-limonene (17.3 µg/ml), is considered to inhibit ACE2-mediated cell entry of another type of coronavirus, HCoV-NL63. The current study examined whether this extract could inhibit the binding of the SARS-CoV-2 spike protein to ACE2. Binding was quantified as cell-bound fluorescence intensity in live cell cultures in which canine kidney MDCK cells overexpressing ACE2 were incubated with fluorescein-labeled S1 spike protein. When indigo extract, together with S1 protein, was added at 8,650x and 17,300x dilutions, fluorescence intensity decreased in a dose- and S1 extract-dependent manner, without affecting cell viability. When 4.0-nM tryptanthrin was added instead of the indigo extract, fluorescence intensity also decreased, but to a lesser degree than with indigo extract. Docking simulation analyses revealed that tryptanthrin readily bound to the receptor-binding domain of the S1 protein, and identified 2- and 7-amino acid sequences as the preferred binding sites. The indigo extract appeared to inhibit S1-ACE2 binding at high dilutions, and evidently contained other inhibitory elements as well as tryptanthrin. This extract may be useful for the prevention or treatment of SARS-CoV-2 infection.

Elevation of intraocular pressure is a major risk factor for glaucoma development, which causes the loss of retinal ganglion cells (RGCs). Lipocalin 2 (Lcn2) is upregulated in glaucomatous retinae; however, whether Lcn2 is directly involved in glaucoma is debated. In this study, retinal explant cultures were subjected to increased water pressure using a two-chamber culture device, and Lcn2 protein levels were examined by immunoblotting. In situ TdT-mediated dUTP nick and labeling (TUNEL) and glial fibrillary acidic protein (GFAP) immunohistochemical assays were performed to assess apoptosis and gliosis, respectively. The neurotoxicity of Lcn2 in the retinal explant culture was determined with exogenous administration of recombinant Lcn2. The Lcn2 protein levels, percentage of TUNEL-positive cells, and GFAP-positive area were significantly higher in retinae cultured under 50 cm H2O pressure loads compared to those cultured under 20 cm H2O. We found that Lcn2 exhibited neurotoxicity in retinae at dose of 1 μg/ml. The negative effects of increased hydrostatic pressure were attenuated by the iron chelator deferoxamine. This is the first report demonstrating the direct upregulation of Lcn2 by elevating hydrostatic pressure. Modulating Lcn2 and iron levels may be a promising therapeutic approach for retinal degeneration.

Background

Lung alveolar epithelial cell (AEC) apoptosis has attracted attention as an early pathogenic event in the development of idiopathic interstitial pneumonia (IIP); however, the causative mechanism remains unclear. Cell adhesion molecule 1 (CADM1) is an AEC adhesion molecule in the immunoglobulin superfamily. It generates a membrane-associated C-terminal fragment, αCTF, through protease-mediated ectodomain shedding, termed α-shedding. Increased CADM1 α-shedding contributes to AEC apoptosis in emphysematous lungs.

Methods

Formalin-fixed, paraffin-embedded lung lobes (n = 39) from 36 autopsied patients with IIP were classified as acute IIP (n = 10), fibrosing-type nonspecific IIP (f-NSIP, n = 10), cryptogenic organizing IIP (n = 9), and usual IIP (n = 10). CADM1 expression in the lung sections was examined by western blotting and compared with control lungs (n = 10). The rate of CADM1 α-shedding was calculated as the relative amount of αCTF to full-length CADM1, and the full-length CADM1 level was estimated per epithelial cell by normalization to cytokeratin 7, a lung epithelial marker. Apoptotic AECs were detected by immunohistochemistry for single-stranded DNA (ssDNA). NCI-H441 and A549 human lung epithelial cells were transfected with small interfering RNA (siRNA) to silence CADM1 expression and analyzed by terminal nucleotide nick end labeling assays.

Results

The rate of CADM1 α-shedding was higher in all IIP subtypes than in the control (P ≤ 0.019), and the full-length CADM1 level was lower in f-NSIP (P = 0.007). The α-shedding rate and full-length CADM1 level were correlated with each other (P = 0.015) and with the proportion of ssDNA-positive AECs (P ≤ 0.024). NCI-H441 cells transfected with siRNA exhibited a 61 % lower rate of expression of full-length CADM1 and a 17-fold increased proportion of apoptotic cells. Similar results were obtained with A549 cells.

Conclusions

CADM1 α-shedding appeared to be increased in all four IIP subtypes and consequently contributed to AEC apoptosis by decreasing the full-length CADM1 level. This mechanism particularly impacted f-NSIP. The molecular mechanism causing AEC apoptosis may be similar between IIP and emphysema.

This mini-review summarizes the protocol we have developed for the analysis of neutral glycosphingolipids (GSLs) by high-performance thin layer chromatography (HPTLC)-mass spectrometry (MS). We also present results obtained using this glycolipidomic approach to study neutral GSLs from mouse kidney, spleen, and small intestine. Finally, we discuss what is required for further development of this method, as well as what is expected for the future of glycolipid biology.

緑内障や水頭症において病的内圧上昇は神経変性の一因と考えられているが、汎用性や再現性の高い神経病態モデルが少ないため実験的証明が困難であり、分子機序は未だ解明されていない。本研究は緑内障における眼圧上昇と網膜神経節細胞変性との因果関係を明確にし、その病態形成の分子基盤を解析することを目的とする。方法として申請者らが独自に開発した細胞培養加圧装置を用いて圧上昇と神経節細胞変性との相関解析を行い、申請者らによる先行研究に基づいて着想した２つの仮説：神経接着分子Cell adhesion molecule 1 (CADM1)の酵素的切断産物の蓄積による神経軸索変性；鉄イオン制御分子Lipocalin 2 (LCN2)の発現上昇による神経細胞死誘導を検証する。本年度はLCN2と網膜変性との関連を中心に研究計画を推進し、ほぼ完了している。本研究報告は内圧上昇によるLCN2発現上昇を実験的に証明した点において重要であり、その結果グリオーシスを伴う網膜神経節細胞死が誘導されることを明らかにし、動物モデルにおいても再現されることを示した。また、これらの神経変性現象が鉄イオンのキレート剤添加によって減弱されることを明らかにすることによって、緑内障の治療薬開発につながる可能性も示唆した。LCN2の発現上昇は緑内障だけでなく、アルツハイマー病やパーキンソン病などの中枢神経系変性疾患においても報告されている。今後はLCN2の発現上昇に至る発現誘導機構や、LCN2高発現によって活性化する因子の探索など細胞内情報伝達経路を解明することによって、LCN2と神経変性との関連をより詳細に明らかにすることが必須であろう。他方で、来年度はもう一つの仮説の検証を重点的に実施する予定であり、既に共同研究において一定の成果を得るなど準備状況は良好である。

CADM1 is an adhesion molecule expressed abundantly in neurons and epithelial cells. We have revealed that ectodomain shedding of CADM1 is occurred in emphysema, interstitial pneumonia and diabetes and that CADM1 shedding leads to cell apoptosis. In this study, to elucidate pathogenic action of CADM1 shedding in neurodegeneration, we investigated the correlation between enteric nerve degeneration and CADM1 shedding in dilated colon with tumor-induced stenosis. CADM1 shedding rate was increased proportionately with colon dilation in association with decrease of ganglion cells and neurites. Using two-chamber cell culture system enable to loading modest static pressure to the cultured cells, we showed that increasing pressure induced CADM1 shedding and the decreases of neurite density and thickness. Exogenous expression of CADM1 shedding product resulted in neurites loss, suggesting its involvement in neurodegeneration.

Cell adhesion molecule 1 (CADM1) is expressed by lung alveolar cells and pancreatic islet cells, and is enzymatically shed at its juxtamembranous ectodomain. In the present study, we examined CADM1 expression in the lung of idiopathic interstitial pneumonia (IIP) and in the pancreata of type 2 diabetes mellitus (T2DM), and found that CADM1 ectodomain shedding increased in both cases. In IIP lungs, the increase of CADM1 shedding resulted in the decrease of full length CADM1 and led alveolar epithelical cell apoptosis, which assumed to be an early pathogenic event in the development of IIP. In T2DM, the CADM1 shedding rates and haemoglobin A1c levels were correlated, suggesting that increased CADM1 shedding in pancreata contributed to blood glucose dysregulation. Interestingly, in those disease, an imbalanced protease activity has been reported. Our studies lead us to identify the protease-mediated shedding of adhesion molecule as an important pathogenic process for tissue degeneration.

Prosaposin (PSAP) is the precursor protein of saposins (SAPs) which are required for the lysosomal hydrolysis of sphingolipids. Although PSAP itselfis known to be secreted into several body fluids, its physiological function is not well understood. PSAP deficient mice (Psap-/-) are born at the much lower frequency suggesting the function of PSAP during the mouse embryogenesis. In this study, we investigated the temporal and spatial expression of PSAP in the wild-type mouse embryo and the phenotype of Psap-/-embryo. Protein levels of PSAP in the mouse embryonic tissues were low at embryonic day (E) 6.5 and increased at E7.5 to 9.5. Immunohistochemical analyses showed high expressions of PSAP in decidua at E7.5 to 9.5 and in spongiotrophoblastic layer of placenta at E10.5. SAPs were highly expressed in the large lysosomes of visceral endoderm of yolk sac. Histopathological examination of Psap-/-embryos indicated the developmental abnormality as early as at around E7.5. Shrunken cells were observed in decidua. The number and the size of large lysosome in visceral endoderm of yolk sac were decreased. These lethal phenotypes of Psap-/-embryos were rescued by maternal overexpression of PSAP. Our findings suggest that PSAP and SAPs play important roles during the early stage of mouse pregnancy, at the site of decidua, yolk sac and placenta.