Axitinib attenuates the progression of liver fibrosis by restoring mitochondrial function.

Liver fibrosis can progress to cirrhosis and hepatocellular carcinoma, which may eventually lead to liver failure and even death. No direct anti-fibrosis drugs are available at present. Axitinib is a new generation of potent multitarget tyrosine kinase receptor inhibitors, but its role in liver fibrosis remains unclear. In this study, a CCl4-induced hepatic fibrosis mouse model and a TGF-ß1-induced hepatic stellate cell model were used to explore the effect and mechanism of axitinib on hepatic fibrosis. Results confirmed that axitinib could alleviate the pathological damage of liver tissue induced by CCl4 and inhibit the production of glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase. It also inhibited collagen and hydroxyproline deposition and the protein expression of Col-1 and α-SMA in CCl4-induced liver fibrosis. In addition, axitinib inhibited the expression of CTGF and α-SMA in TGF-ß1-induced hepatic stellate cells. Further studies showed that axitinib inhibited mitochondrial damage and reduced oxidative stress and NLRP3 maturation. The use of rotenone and antimycin A confirmed that axitinib could restore the activity of mitochondrial complexes I and III, thereby inhibiting the maturation of NLRP3. In summary, axitinib inhibits the activation of HSCs by enhancing the activity of mitochondrial complexes I and III, thereby alleviating the progression of liver fibrosis. This study reveals the strong potential of axitinib in the treatment of liver fibrosis.

FOXO3 regulates Smad3 and Smad7 through SPON1 circular RNA to inhibit idiopathic pulmonary fibrosis.

Forkhead box protein O3 (FOXO3) has good inhibition ability toward fibroblast activation and extracellular matrix, especially for the treatment of idiopathic pulmonary fibrosis. How FOXO3 regulates pulmonary fibrosis remains unclear. In this study, we reported that FOXO3 had binding sequences with F-spondin 1 (SPON1) promoter, which can activate its transcription and selectively promote the expression of SPON1 circRNA (circSPON1) but not mRNA expression. We further demonstrated that circSPON1 was involved in the extracellular matrix deposition of HFL1. In the cytoplasm, circSPON1 directly interacted with TGF-ß1-induced Smad3 and inhibited the activation of fibroblasts by inhibiting nuclear translocation. Moreover, circSPON1 bound to miR-942-5p and miR-520f-3p that interfered with Smad7 mRNA and promoted Smad7 expression. This study revealed the mechanism of FOXO3-regulated circSPON1 in the development of pulmonary fibrosis. Potential therapeutic targets and new insights into the diagnosis and treatment of idiopathic pulmonary fibrosis based on circRNA were also provided.

Clinical and Basic Evaluation of the Effects of Upregulated TNFAIP3 Expression on Colorectal Cancer.

Objective: To assess the TNFAIP3 and nuclear factor κB (NFκB) protein expressions in colorectal cancer (CRC) tissue and to analyze the association of these proteins with the clinical pathological characteristics of CRC. Methods: The following methods should be used in clinical trials: information collection and immunohistochemical methods. The following methods are used for cell experiment: cell transfection, CCK8 detection method, transwell experiment, and western blot experiment. Explore the TNFAIP3 expression in CRC cells, and assess the effect of upregulated TNFAIP3 expression on CRC cell proliferation, invasion, and migration. In clinical experiment, we selected the tumor tissues of 39 CRC patients as our experimental samples. We also collected corresponding patient demographics, such as sex, age, cell differentiation, tumor type, and lymph node metastasis. We also analyzed the TNFAIP3 and NFκB protein expressions in 20 experimental and 20 control samples and evaluated potential correlations between these two proteins and clinical pathological characteristics of CRC. For basic experiment, we established CRC cell lines with elevated TNFAIP3 expression and then randomly divided the cells into three groups, namely, TNFAIP3, NS, and Con groups. Using the transwell and CCK8 methods, we detected the CRC migration abilities and cell proliferation, respectively. We also employed western blot analysis to assess protein expression in the three groups. Results: NFκB was highly expressed, and TNFAIP3 was scarcely expressed in the experimental group versus control. The expression of both these proteins were strongly related to the degree of tumor differentiation (P < 0.05). The TNFAIP3 and NFκB protein expressions were significantly associated with lymph node metastasis and tumor differentiation (P < 0.05). For basic experiment, compared to the Con and NS groups, TNFAIP3 protein expression levels, cell proliferation, invasion, and migration were significantly increased in the TNFAIP3 group (P < 0.05). Conclusion: TNFAIP3 overexpression strongly inhibited CRC proliferation, invasion, and migration. Enhanced NFκB protein expression in CRC tissues was associated with elevated malignant degree, metastasis, and TNFAIP3 protein expression in patients who demonstrated high malignant degree and metastasis. Our evidences suggest the promising potential of utilizing TNFAIP3 and NFκB as important reference indices for determining the prognostic outcome of CRC. Furthermore, we revealed that TNFAIP3 overexpression inhibited CRC cell proliferation, invasion, and migration.

Inhibitory effect of Idelalisib on selenite-induced cataract in Sprague Dawley rat pups.

PURPOSE: The aim of this study was to evaluate the therapeutic effect of Idelalisib, Apelisib and Copanlisib on 8-day-old cataract SD rat pups. MATERIALS AND METHODS: The rat model induced by sodium selenate (Na2SeO3) was used in this study. Experimental animals were randomly divided into five groups with eight animals in each group. They were control group, Na2SeO3 group, Idelalisib group, Apelisib group and Copanlisib group. On days 3, 5 and 7, all rats in Na2SeO3, Idelalisib, Apelisib and Copanlisib groups were given subcutaneous injection into the nape with Na2SeO3 and control group was given the same amount of saline. For days 1-14, Idelalisib, Apelisib and Copanlisib were given by intragastric administration, respectively, and the same amount of saline was given to the control group and Na2SeO3 group. On the 15th day of the experiment, we selected the Idelalisib group with the best effect from all groups, separated their lenses, and further analyzed the crystal proteins, oxidative damage and apoptosis indexes. RESULTS: The survival rate of rats in control and Idelalisib groups was 100%, the Na2SeO3 group was 37.5%, the Apelisib group was 25%, and the Copanlisib group was 0. According to the rat survival rate and lens score, we selected Idelalisib for further analysis of the crystallin, oxidative damage and apoptosis indexes. The results suggested that Na2SeO3 leads to cataract formation and crystallin precipitation. The levels of antioxidant enzymes GSH and SOD were decreased in the Na2SeO3 group, Nrf-2 and HO-1 were downregulated, Keap1 upregulated, and cleaved caspase-3 and Bax/Bcl-2 upregulated. Idelalisib significantly improved crystallin insolubility, reduced oxidative damage, and inhibited lens apoptosis. CONCLUSION: In summary, Idelalisib can significantly improve the progression of Na2SeO3-induced cataract in rats. In the future, it may be a potential effective drug candidate for the clinical treatment of cataract.

Heterologous prime-boost immunizations with chimpanzee adenoviral vectors elicit potent and protective immunity against SARS-CoV-2 infection.

A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is urgently needed to tackle the COVID-19 global pandemic. Here, we describe the development of chimpanzee adenovirus serotypes 6 and 68 (AdC6 and AdC68) vector-based vaccine candidates expressing the full-length transmembrane spike glycoprotein. We assessed the vaccine immunogenicity, protective efficacy, and immune cell profiles using single-cell RNA sequencing in mice. Mice were vaccinated via the intramuscular route with the two vaccine candidates using prime-only regimens or heterologous prime-boost regimens. Both chimpanzee adenovirus-based vaccines elicited strong and long-term antibody and T cell responses, balanced Th1/Th2 cell responses, robust germinal center responses, and provided effective protection against SARS-CoV-2 infection in mouse lungs. Strikingly, we found that heterologous prime-boost immunization induced higher titers of protective antibodies, and more spike-specific memory CD8+ T cells in mice. Potent neutralizing antibodies produced against the highly transmissible SARS-CoV-2 variants B.1.1.7 lineage (also known as N501Y.V1) and B.1.351 lineage (also known as N501Y.V2) were detectable in mouse sera over 6 months after prime immunization. Our results demonstrate that the heterologous prime-boost strategy with chimpanzee adenovirus-based vaccines is promising for further development to prevent SARS-CoV-2 infection.

Effects of Upregulation of TNFAIP3 on Diabetic Neuropathic Pain in Mice.

Globally, diabetes has assumed epidemic proportions with the neuropathic complications attributed to the malady emerging as a substantial burden on patients and society. DNP has greatly affected the daily life of patients, the effect of traditional treatment methods is not ideal, and it is easy to produce drug resistance. This work is aimed at scrutinizing the effect of upregulating the expression of TNFAIP3 on diabetic neuralgia in mice. This work entailed ascertaining the effects of TNFAIP3 on a murine DNP system. This inspired us to observe the analgesic effect via high expression of lentivirus-mediated TNFAIP3 by intrathecal injection in the animal model to explore its regulatory impacts, symptom relief, and mechanistic role in pain. The results displayed an attenuation of hind paw pain hypersensitivity by LV-TNFAIP3 in the animals. The spinal cord and dorsal root ganglion of mice with neuropathic pain displayed an evident dip in TNFAIP3. Inhibition of the ERK/NF-κB signaling pathway employing LV-TNFAIP3 conspicuously suppressed this pathway while the diabetic pain hypersensitivity was quelled. This effect was also seen with insulin treatment evidently. In conclusion, according to the above analyses, the interaction between DNP and extracellular signal-regulated kinase signal transduction pathway is one of the key factors of pathogenesis.

Protective effect of Idelalisib on carbon tetrachloride-induced liver fibrosis via microRNA-124-3P/phosphatidylinositol-3-hydroxykinase signalling pathway.

Liver fibrosis is the repair process of abnormal connective tissue hyperplasia after liver damage caused by different causes. Inhibition of PI3K/Akt signalling pathway can reduce the deposition of extracellular matrix, inhibit the proliferation of hepatic stellate cells (HSCs), and promote its apoptosis to achieve the purpose of therapy. This study aimed to investigate the effect of Idelalisib (PI3K inhibitor) on carbon tetrachloride (CCl4 )-induced liver fibrosis in mice. We used CCl4 -induced liver fibrosis mouse model in vivo and TGF-ß1-stimulated HSCs to evaluate the antifibrosis activity of Idelalisib. In vivo, Idelalisib significantly alleviated CCl4 -induced liver damage, collagen deposition, and hydroxyproline accumulation in mice. Immunohistochemistry and Western blot results showed that Idelalisib could significantly inhibit the expressions of COL1 and α-SMA in a concentration-dependent manner. In cell experiments, Idelalisib significantly inhibited the expressions of COL1, SMA, and p-Smad3 in TGF-ß-induced HSCs, thereby inhibiting HSC activation. Flow cytometry and Western blot results showed that Idelalisib significantly promoted TGFß-induced apoptosis of HSCs after 48 h of administration, but had no significant effect after 24 h. Idelalisib promoted the apoptosis of activated HSCs by inhibiting the PI3K/Akt/FOXO3 signalling pathway. To further explore the mechanism by which Idelalisib inhibited PI3K, we predicted the miRNA targeting PI3K through the database and crossed it with the down-regulated miRNA reported in liver fibrosis mice in the past five years. Finally, we identified miR-124-3p and miR-143-3p. We then demonstrated that Idelalisib significantly promoted miR-124-3p and miR-142-3p in vitro and in vivo. Dual-luciferase report analysis showed that Idelalisib significantly inhibited luciferase activity but had no significant effect on the luc-MUT transfection assay. Finally, we demonstrated that Idelalisib reversed the effects of miR-124-3p inhibitor on the PI3K/Akt/FOXO3 asterisk pathway and caspase-3. Idelalisib has potential as a candidate drug for alleviating liver fibrosis.

Validation of novel hub genes and molecular mechanisms in acute lung injury using an integrative bioinformatics approach.

Acute lung injury (ALI) is an inflammatory pulmonary disease that can easily develop into serious acute respiratory distress syndrome, which has high morbidity and mortality. However, the molecular mechanism of ALI remains unclear, and few molecular biomarkers for diagnosis and treatment have been identified. In this study, we aimed to identify novel molecular biomarkers using a bioinformatics approach. Gene expression data were obtained from the Gene Expression Omnibus database, co-expressed differentially expressed genes (CoDEGs) were identified using R software, and further functional enrichment analyses were conducted using the online tool Database for Annotation, Visualization, and Integrated Discovery. A protein-protein interaction network was established using the STRING database and Cytoscape software. Lipopolysaccharide (LPS)-induced ALI mouse model was constructed and verified. The hub genes were screened and validated in vivo. The transcription factors (TFs) and miRNAs associated with the hub genes were predicted using the NetworkAnalyst database. In total, 71 CoDEGs were screened and found to be mainly involved in the cytokine-cytokine receptor interactions, and the tumor necrosis factor and malaria signaling pathways. Animal experiments showed that the lung injury score, bronchoalveolar lavage fluid protein concentration, and wet-to-dry weight ratio were higher in the LPS group than those in the control group. Real-time polymerase chain reaction analysis indicated that most of the hub genes such as colony-stimulating factor 2 (Csf2) were overexpressed in the LPS group. A total of 20 TFs including nuclear respiratory factor 1 (NRF1) and two miRNAs were predicted to be regulators of the hub genes. In summary, Csf2 may serve as a novel diagnostic and therapeutic target for ALI. NRF1 and mmu-mir-122-5p may be key regulators in the development of ALI.

Reducing inflammation and rescuing FTD-related behavioral deficits in progranulin-deficient mice with α7 nicotinic acetylcholine receptor agonists.

Mutations in the progranulin gene cause frontotemporal dementia (FTD), a debilitating neurodegenerative disease that involves atrophy of the frontal and temporal lobes and affects personality, behavior, and language. Progranulin-deficient mouse models of FTD exhibit deficits in compulsive and social behaviors reminiscent of patients with FTD, and develop excessive microgliosis and increased release of inflammatory cytokines. Activation of nicotinic acetylcholine receptors (nAChRs) by nicotine or specific α7 nAChR agonists reduces neuroinflammation. Here, we investigated whether activation of nAChRs by nicotine or α7 agonists improved the excessive inflammatory and behavioral phenotypes of a progranulin-deficient FTD mouse model. We found that treatment with selective α7 agonists, PHA-568487 or ABT-107, strongly suppressed the activation of NF-κB in progranulin-deficient cells. Treatment with ABT-107 also reduced microgliosis, decreased TNFα levels, and reduced compulsive behavior in progranulin-deficient mice. Collectively, these data suggest that targeting activation of the α7 nAChR pathway may be beneficial in decreasing neuroinflammation and reversing some of the behavioral deficits observed in progranulin-deficient FTD.

[Effects of hypertonic sodium chloride hydroxyethyl starch solution on cerebral vasospasm following subarachnoid hemorrhage and its mechanism].

OBJECTIVE: To investigate the protective effect and potential mechanisms of hypertonic sodium chloride hydroxyethyl starch solution (HSH) against the cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH). METHODS: Twenty-four male Sprague-Dawley (SD) rats were randomly assigned to four groups according to the random number table, with 6 rats in each group. The SAH-CVS model was reproduced by injection of the blood twice through the cisterna magna. Rats in both model and HSH treatment groups received 8 mL/kg normal saline (NS) or HSH treatment everyday via caudal vein. Rats in sham group were injected with 1.5 mL/kg NS into cisterna magna followed by 8 mL/kg NS treatment. Rats in normal group received no treatment. Rats were sacrificed to harvest basilar artery after 7 days. The thickness of vessel wall and lumen area were measured using hematoxylin-eosin (HE) staining. The rate of apoptosis of vascular smooth muscle cell (VSMC) was assessed using flow cytometry. Caspase-3 activity was measured by a fluorometric assay. The expressions of Bax and Bcl-2 were determined by Western Blot. Intracellular reactive oxygen species (ROS) was detected by H2DCFDA. RESULTS: Compared with normal group, increased thickness of vessel wall (27.72 ± 1.94 µm vs. 18.30 ± 1.10 µm, P<0.05), decreased lumen area (26 115 ± 1 991 µm² vs. 55 080 ± 2 091 µm², P<0.05), and elevation of rate of apoptosis of VSMCs [(35.05 ± 5.54) % vs. (5.93 ± 1.53) %, P<0.05] were found in model group. Compared with model group, decreased thickness of vessel wall (22.55 ± 1.50 µm vs. 27.72 ± 1.94 µm, P<0.05), increase of lumen area (48 115 ± 2 460 µm² vs. 26 115 ± 1 991 µm², P<0.05), and depressed rate of apoptosis of VSMCs [(16.54 ± 5.94) % vs. (35.05 ± 5.54) %, P<0.05] were found in HSH treatment group. Caspase-3 activity, intracellular ROS level, Bax and Bcl-2 expressions in model group were (188.40 ± 19.35)%, (163.50 ± 17.02)%, (208.71 ± 26.04)% and (44.52 ± 9.61) % of those of normal group, and the differences of these parameters between model and normal groups were statistically significant (all P<0.05). Caspase-3 activity, intracellular ROS level, Bax and Bcl-2 expressions in HSH treatment group were (135.05 ± 19.52)%, (119.44 ± 11.50)%, (139.20 ± 18.04)% and (85.35 ± 13.12)% of those of normal group, respectively, and the differences of these parameters between HSH treatment and model groups were statistically significant (all P<0.05). The differences of all measurements between sham and normal groups were not statistically significant. CONCLUSIONS: The current results demonstrate that HSH attenuates the SAH-induced CVS, alleviates thickness of vessel wall, and increases lumen area via inhibition of VSMCs apoptosis.

Cerebrospinal fluid cytokine dynamics differ between Alzheimer disease patients and elderly controls.

The time courses of levels of multiple plasma and cerebrospinal fluid (CSF) cytokines in patients with Alzheimer disease (AD) and in age-matched control subjects were compared. Interleukin (IL)-1ß, IL-2, IL-6, IL-8, IL-10, IL-12p70, granulocyte-macrophage colony-stimulating factor, interferon-γ, and tumor necrosis factor alpha levels were measured 7 times over a 24-hour period in plasma and CSF using a lumbar catheter. Baseline plasma and CSF cytokine levels were found to be similar in AD and control subjects. However, the CSF levels of all measured cytokines, except IL-6 and IL-8, diverged over time between AD and control subjects, such that CSF cytokine levels in AD subjects were higher than in controls. This difference was greatest at 24 hours after the insertion of the lumbar catheter. In contrast, no differences in cytokine trajectories were seen in plasma. These data suggest that the neuroinflammatory response to lumbar catheter placement differs between AD and control subjects.

Role of α7 nicotinic acetylcholine receptors in regulating tumor necrosis factor-α (TNF-α) as revealed by subtype selective agonists.

Immunological responses to protect against excessive inflammation can be regulated by the central nervous system through the cholinergic anti-inflammatory pathway wherein acetylcholine released from vagus nerves can inhibit inflammatory cytokines. Although a role for the α7 nicotinic acetylcholine receptor (α7 nAChR) in mediating this pathway has been suggested, pharmacological modulation of the pathway by selective agonists remains to be further elucidated. In this study, the role of α7 nAChRs in the regulation of TNF-α release was investigated using high affinity and selective α7 nAChR agonists in mouse peritoneal macrophage and human whole blood in vitro, and in mouse serum in vivo. In mouse peritoneal macrophages, LPS-induced TNF-α release in vitro was inhibited by a selective α7 nAChR agonist, A-833834 (5-[6-(5-Methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyridazin-3-yl]-1H-indole), and that effect was attenuated by α7 nAChR antagonist methyllycaconitine. The inhibitory effect of A-833834 on LPS-induced TNF-α release was also observed in human whole blood in vitro. I.v. LPS-induced TNF-α release in mouse serum was attenuated following i.p. administration of A-833834. Similarly, i.v. LPS-induced TNF-α release in mouse serum was also attenuated following i.p. administration of A-585539, another α7 nAChR agonist with limited brain penetration, suggesting that these effects are mediated by peripheral α7 nAChRs. A-833834 was also efficacious in suppressing TNF-α release in mouse serum following oral administration in zymosan-induced peritonitis. These studies collectively demonstrate that selectively targeting α7 nAChRs could offer a novel therapeutic modality to treat acute and chronic inflammatory disease states.

In vitro pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107.

Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity alpha7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

Alpha7 nAChR-mediated activation of MAP kinase pathways in PC12 cells.

The alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) plays a fundamental role in Ca(2+)-dependent activation of signaling pathways that can modulate intracellular events involved in learning and memory. Activation of extracellular signal-regulated kinase-1 and -2 (ERK1/2) are well documented Ca(2+) signaling events, but these have not been well characterized in response to alpha7 nAChR-selective ligands. The present study examined activation of ERK1/2 and explored pathways leading to CREB phosphorylation utilizing alpha7 nAChR-selective ligands in PC12 cells endogenously expressing alpha7 nAChRs. Robust concentration-dependent increase in ERK1/2 phosphorylation was triggered by structurally diverse alpha7 nAChR agonists such as nicotine, choline, GTS-21, SSR-180711A and PNU-282987 in the presence of the positive allosteric modulator (PAM) PNU-120596. This effect was attenuated by selective alpha7 nAChR antagonists or by chelation of extracellular Ca(2+). ERK1/2 phosphorylation was also attenuated by inhibitors of calmodulin-dependent protein kinase II (CaMKII), p38 MAP kinase and mitogen-activated protein kinase kinase1/2 (MEK1/2), indicating the involvement of these kinases upstream of ERK1/2. This was confirmed by direct measurement of p38 MAPK and MEK1/2 phosphorylation. These data suggest that alpha7 nAChR agonist-triggered Ca(2+) transient in PC12 cells induces activation of CaMKII, leading to sequential phosphorylation of p38 MAPK, MEK1/2, ERK1/2 and CREB. Such mechanisms may endow the alpha7 nAChRs with roles in modulating Ca(2+)-dependent intracellular second messenger events implicated in diverse aspects of cognition.

Positive allosteric modulation of alpha7 neuronal nicotinic acetylcholine receptors: lack of cytotoxicity in PC12 cells and rat primary cortical neurons.

BACKGROUND AND PURPOSE: alpha7-Nicotinic acetylcholine receptors (alpha7 nAChRs) play an important role in cognitive function. Positive allosteric modulators (PAMs) amplify effects of alpha7 nAChR agonist and could provide an approach for treatment of cognitive deficits in neuropsychiatric diseases. PAMs can either predominantly affect the apparent peak current response (type I) or increase both the apparent peak current response and duration of channel opening, due to prolonged desensitization (type II). The delay of receptor desensitization by type II PAMs raises the possibility of Ca2+-induced toxicity through prolonged activation of alpha7 nAChRs. The present study addresses whether type I and II PAMs exhibit different cytotoxicity profiles. EXPERIMENTAL APPROACH: The present studies evaluated cytotoxic effects of type I PAM [N-(4-chlorophenyl)]-alpha-[(4-chlorophenyl)-aminomethylene]-3-methyl-5-isoxazoleacet-amide (CCMI) and type II PAM 1-[5-chloro-2,4-dimethoxy-phenyl]-3-[5-methyl-isoxazol-3-yl]-urea (PNU-120596), or 4-[5-(4chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulphonamide (A-867744). The studies used cultures of PC12 cells and primary cultures of rat cortical neuronal cells. KEY RESULTS: Our results showed that neither type I nor type II PAMs had any detrimental effect on cell integrity or cell viability. In particular, type II PAMs did not affect neuron number and neurite outgrowth under conditions when alpha7 nAChR activity was measured by Ca2+ influx and extracellular signal-regulated kinases 1 and 2 phosphorylation, following exposure to alpha7 nAChR agonists. CONCLUSIONS AND IMPLICATIONS: This study demonstrated that both type I and type II alpha7 nAChR selective PAMs, although exhibiting differential electrophysiological profiles, did not exert cytotoxic effects in cells endogenously expressing alpha7 nAChRs.

Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells.

Beta-amyloid peptide 1-42 (Abeta(1-42)) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Abeta(1-42) can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Abeta(1-42)-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Abeta(1-42) increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Abeta(1-42)-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at tyrosine-216 (GSK-3beta-pY216), which was partially inhibited by the GSK-3beta inhibitor, CHIR98023. Abeta(1-42)-induced tau phosphorylation and increase in GSK-3beta-pY216 phosphorylation were also partially attenuated by alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and alpha-bungarotoxin. The alpha7 nAChR agonist and the GSK-3beta inhibitor had no additive effect. These observations suggest that alpha7 nAChR modulation can influence Abeta(1-42)-induced tau phosphorylation, possibly involving GSK-3beta. This study provides evidence of nAChR mechanisms underlying Abeta(1-42) toxicity and tau phosphorylation, which, if translated in vivo, could provide additional basis for the utility of alpha7 nAChR ligands in the treatment of Alzheimer's disease.

Analysis of gene expression profiles in rat hippocampus following treatment with nicotine and an alpha7 nAChR selective agonist.

The nicotinic acetylcholine receptors (nAChRs) play critical roles in neuronal transmission and modulation. Among the diverse nAChRs, the alpha7 subtype has been considered as a potential therapeutic target for treating cognitive deficits associated with neuropsychiatric and neurodegenerative diseases. Although a number of mechanisms including neurotransmitter and biochemical effects linking alpha7 nAChR activation and cognitive function are beginning to be described, the underlying molecular processes especially following repeated administration remain unclear. To address this, we have performed gene expression analysis in rats treated with nicotine and a selective alpha7 nAChR agonist, PNU-282987. Our results showed significant overlap in gene expression changes induced by PNU-282987 and nicotine, suggesting convergent pathways triggered by these compounds. Treatment with nicotine also resulted in regulation of a number of genes that were not regulated by PNU-282987, consistent with the interaction of nicotine with other nAChRs beyond the alpha7 subtype. Interestingly, these gene expression changes were observed 24 h post-dose, suggesting that both nicotine and PNU-282987 cause protracted changes in gene expression. Overall, our results identify gene expression changes that may contribute to further defining the roles of nAChR activation in cognitive function.

Identification of a silicatein(-related) protease in the giant spicules of the deep-sea hexactinellid Monorhaphis chuni.

Silicateins, members of the cathepsin L family, are enzymes that have been shown to be involved in the biosynthesis/condensation of biosilica in spicules from Demospongiae (phylum Porifera), e.g. Tethya aurantium and Suberites domuncula. The class Hexactinellida also forms spicules from this inorganic material. This class of sponges includes species that form the largest biogenic silica structures on earth. The giant basal spicules from the hexactinellids Monorhaphis chuni and Monorhaphis intermedia can reach lengths of up to 3 m and diameters of 10 mm. The giant spicules as well as the tauactines consist of a biosilica shell that surrounds the axial canal, which harbours the axial filament, in regular concentric, lamellar layers, suggesting an appositional growth of the spicules. The lamellae contain 27 kDa proteins, which undergo post-translational modification (phosphorylation), while total spicule extracts contain additional 70 kDa proteins. The 27 kDa proteins cross-reacted with anti-silicatein antibodies. The extracts of spicules from the hexactinellid Monorhaphis displayed proteolytic activity like the silicateins from the demosponge S. domuncula. Since the proteolytic activity in spicule extracts from both classes of sponge could be sensitively inhibited by E-64 (a specific cysteine proteinase inhibitor), we used a labelled E-64 sample as a probe to identify the protein that bound to this inhibitor on a blot. The experiments revealed that the labelled E-64 selectively recognized the 27 kDa protein. Our data strongly suggest that silicatein(-related) molecules are also present in Hexactinellida. These new results are considered to also be of impact for applied biotechnological studies.

Positive and negative regulation of the gamma-secretase activity by nicastrin in a murine model.

Nicastrin is a component of the gamma-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrin-deficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid beta-peptide and failed to release the intracellular domain of APP- or Notch1-Gal4-VP16 fusion proteins. Additionally, C- and N-terminal fragments of PS1 and the C-terminal fragments of PS2 were not detectable in Nicastrin-null fibroblasts, whereas full-length PS1 accumulated in null fibroblasts, indicating that Nicastrin is required for the endoproteolytic processing of presenilins. Interestingly, cells derived from Nicastrin heterozygotes produced relatively higher levels of amyloid beta-peptide whether the source was endogenous mouse or transfected human APP. These data demonstrate that Nicastrin is essential for the gamma-secretase cleavage of APP and Notch in mammalian cells and that Nicastrin has both positive and negative functions in the regulation of gamma-secretase activity.