Agomelatine improves memory and learning impairments in a rat model of LPS-induced neurotoxicity by modulating the ERK/SorLA/BDNF/TrkB pathway.

The mutual interplay between neuroinflammation, synaptic plasticity, and autophagy has piqued researchers' interest, particularly when it comes to linking their impact and relationship to cognitive deficits. Being able to reduce inflammation and apoptosis, melatonin has shown to have positive neuroprotective effects; that is why we thought to check the possible role of agomelatine (AGO) as a promising candidate that could have a positive impact on cognitive deficits. In the current study, AGO (40 mg/kg/day, p.o., 7 days) successfully ameliorated the cognitive and learning disabilities caused by lipopolysaccharide (LPS) in rats (250 µg/kg/day, i.p., 7 days). This positive impact was supported by improved histopathological findings and improved spatial memory as assessed using Morris water maze. AGO showed a strong ability to control BACE1 activity and to rein in the hippocampal amyloid beta (Aß) deposition. Also, it improved neuronal survival, neuroplasticity, and neurogenesis by boosting BDNF levels and promoting its advantageous effects and by reinforcing the pTrkB expression. In addition, it upregulated the pre- and postsynaptic neuroplasticity biomarkers resembled in synapsin I, synaptophysin, and PSD-95. Furthermore, AGO showed a modulatory action on Sortilin-related receptor with A-type repeats (SorLA) pathway and adjusted autophagy. It is noteworthy that all of these actions were abolished by administering PD98059 a MEK/ERK pathway inhibitor (0.3 mg/kg/day, i.p., 7 days). In conclusion, AGO administration significantly improves memory and learning disabilities associated with LPS administration by modulating the ERK/SorLA/BDNF/TrkB signaling pathway parallel to its capacity to adjust the autophagic process.

Inhibition of NLRP1 inflammasome improves autophagy dysfunction and Aß disposition in APP/PS1 mice.

Increasing evidence has shown that the NOD-like receptor protein 1 (NLRP1) inflammasome is associated with Aß generation and deposition, which contributes to neuronal damage and neuronal-inflammation in Alzheimer's disease (AD). However, the specific mechanism of NLRP1 inflammasome in the pathogenesis of AD is still unclear. It has been reported that autophagy dysfunction can aggravate the pathological symptoms of AD and plays an important role in regulating Aß generation and clearance. We hypothesized that NLRP1 inflammasome activation may induce autophagy dysfunction contributing to the progression of AD. In the present study, we observed the relationship between Aß generation and NLRP1 inflammasome activation, as well as AMPK/mTOR mediated-autophagy dysfunction in WT 9-month-old (M) mice, APP/PS1 6 M and APP/PS1 9 M mice. Additionally, we further studied the effect of NLRP1 knockdown on cognitive function, Aß generation, neuroinflammation and AMPK/mTOR mediated autophagy in APP/PS1 9 M mice. Our results indicated that NLRP1 inflammasome activation and AMPK/mTOR mediated-autophagy dysfunction are closely implicated in Aß generation and deposition in APP/PS1 9 M mice, but not in APP/PS1 6 M mice. Meanwhile, we found that knockdown of NLRP1 significantly improved learning and memory impairments, decreased the expressions of NLRP1, ASC, caspase-1, p-NF-κB, IL-1ß, APP, CTF-ß, BACE1 and Aß1-42, and decreased the level of p-AMPK, Beclin 1 and LC3 II, and increased the level of p-mTOR and P62 in APP/PS1 9 M mice. Our study suggested that inhibition of NLRP1 inflammasome activation improves AMPK/mTOR mediated-autophagy dysfunction, resulting in the decrease of Aß generation, and NLRP1 and autophagy might be important targets to delay the progression of AD.

Gamma secretase activity modulates BMP-7-induced dendritic growth in primary rat sympathetic neurons.

Autonomic dysfunction has been observed in Alzheimer's disease (AD); however, the effects of genes involved in AD on the peripheral nervous system are not well understood. Previous studies have shown that presenilin-1 (PSEN1), the catalytic subunit of the gamma secretase (γ-secretase) complex, mutations in which are associated with familial AD function, regulates dendritic growth in hippocampal neurons. In this study, we examined whether the γ-secretase pathway also influences dendritic growth in primary sympathetic neurons. Using immunoblotting and immunocytochemistry, molecules of the γ-secretase complex, PSEN1, PSEN2, PEN2, nicastrin and APH1a, were detected in sympathetic neurons dissociated from embryonic (E20/21) rat sympathetic ganglia. Addition of bone morphogenetic protein-7 (BMP-7), which induces dendrites in these neurons, did not alter expression or localization of γ-secretase complex proteins. BMP-7-induced dendritic growth was inhibited by siRNA knockdown of PSEN1 and by three γ-secretase inhibitors, γ-secretase inhibitor IX (DAPT), LY-411575 and BMS-299897. These effects were specific to dendrites and concentration-dependent and did not alter early downstream pathways of BMP signaling. In summary, our results indicate that γ-secretase activity enhances BMP-7 induced dendritic growth in sympathetic neurons. These findings provide insight into the normal cellular role of the γ-secretase complex in sympathetic neurons.

Vitamin D alleviates cognitive dysfunction and brain damage induced by copper sulfate intake in experimental rats: focus on its combination with donepezil.

This study aimed to demonstrate the potential benefits of donepezil (DPZ) and vitamin D (Vit D) in combination to counteract the neurodegenerative disorders induced by CuSO4 intake in experimental rats. Neurodegeneration (Alzheimer-like) was induced in twenty-four male Wistar albino rats by CuSO4 supplement to drinking water (10 mg/L) for 14 weeks. AD rats were divided into four groups: untreated AD group (Cu-AD) and three treated AD groups; orally treated for 4 weeks with either DPZ (10 mg/kg/day), Vit D (500 IU/kg/day), or DPZ + Vit D starting from the 10th week of CuSO4 intake. Another six rats were used as normal control (NC) group. The hippocampal tissue content of ß-amyloid precursor protein cleaving enzyme 1 (BACE1), phosphorylated Tau (p-tau), clusterin (CLU), tumor necrosis factor-α (TNF-α), caspase-9 (CAS-9), Bax, and Bcl-2 and the cortical content of acetylcholine (Ach), acetylcholinesterase (AChE), total antioxidant capacity (TAC), and malondialdehyde (MDA) were measured. Cognitive function tests (Y-maze) and histopathology studies (hematoxylin and eosin and Congo red stains) and immunohistochemistry for neurofilament. Vit D supplementation alleviated CuSO4-induced memory deficits including significant reduction hippocampal BACE1, p-tau, CLU, CAS-9, Bax, and TNF-α and cortical AChE and MDA. Vit D remarkably increased cortical Ach, TAC, and hippocampal Bcl-2. It also improved neurobehavioral and histological abnormalities. The effects attained by Vit D treatment were better than those attained by DPZ. Furthermore, Vit D boosted the therapeutic potential of DPZ in almost all AD associated behavioral and pathological changes. Vit D is suggested as a potential therapy to retard neurodegeneration.

Hydroxysafflor Yellow A Exerts Neuroprotective Effect by Reducing Aß Toxicity Through Inhibiting Endoplasmic Reticulum Stress in Oxygen-Glucose Deprivation/Reperfusion Cell Model.

Ischemia stroke is thought to be one of the vascular risks associated with neurodegenerative diseases, such as Alzheimer's disease (AD). Hydroxysafflor yellow A (HSYA) has been reported to protect against stroke and AD, while the underlying mechanism remains unclear. In this study, SH-SY5Y cell model treated with oxygen-glucose deprivation/reperfusion (OGD/R) was used to explore the potential mechanism of HSYA. Results from cell counting kit-8 (CCK-8) showed that 10 µM HSYA restored the cell viability after OGD 2 hours/R 24 hours. HSYA reduced the levels of malondialdehyde and reactive oxygen species, while improved the levels of superoxide dismutase and glutathione peroxidase. Furthermore, apoptosis was inhibited, and the expression of brain-derived neurotrophic factor was improved after HSYA treatment. In addition, the expression levels of amyloid-ß peptides (Aß) and BACE1 were decreased by HSYA, as well as the expression levels of binding immunoglobulin heavy chain protein, PKR-like endoplasmic reticulum (ER) kinase pathway, and activating transcription factor 6 pathway, whereas the expression level of protein disulfide isomerase was increased. Based on these results, HSYA might reduce Aß toxicity after OGD/R by interfering with apoptosis, oxidation, and neurotrophic factors, as well as relieving ER stress.

Insulin Signaling Disruption and INF-γ Upregulation Induce Aß1-42 and Hyperphosphorylated-Tau Proteins Synthesis and Cell Death after Paraquat Treatment of Primary Hippocampal Cells.

Acute and long-term paraquat (PQ) exposure produces hippocampal neurodegeneration and cognition decline. Although some mechanisms involved in these effects were found, the rest are unknown. PQ treatment, for 1 and 14 days, upregulated interferon-gamma signaling, which reduced insulin levels and downregulated the insulin pathway through phosphorylated-c-Jun N-terminal-kinase upregulation, increasing glucose levels and the production of Aß1-42 and phosphorylated-tau, by beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) overexpression and phosphorylated-GSK3ß (p-GSK3ß; ser9) level reduction, respectively, which induced primary hippocampal neuronal loss. This novel information on the PQ mechanisms leading to hippocampal neurodegeneration could help reveal the PQ actions that lead to cognition dysfunction.

Melatonin Attenuates Vascular Smooth Muscle Contraction Through the γ-Secretase/Notch Intracellular Domain/Myocardin Pathway.

ABSTRACT: Inositol 1, 4, 5-trisphosphate (IP3) signaling-mediated calcium release drives the contraction of vascular smooth muscles and hence regulates blood vessel volume and blood pressure. Melatonin supplementation has been suggested to be beneficial for hypertension. To determine whether the blood pressure-lowering effect of melatonin was accounted for by IP3 signaling, we evaluated the vasoconstriction response and IP3 signaling in isolated mouse thoracic aortic rings during melatonin incubation. C57BL/6 mice were given intraperitoneal injections daily with melatonin, and the systolic blood pressure and contractility of aortic rings from melatonin-treated mice were decreased, and the contraction suppression effect of melatonin was attributed to the impaired expression of contractile proteins in vascular smooth muscle cells rather than IP3 signaling. Our results further showed that melatonin increased the expression of γ-secretase, which could cleave and release the notch intracellular domain, and the notch intracellular domain prevented the transcription of contractile genes by interfering with the interaction between serum response factor and myocardin, the master regulator of contractile protein. In this article, we report a novel mechanism by which melatonin regulates smooth muscle contractility that does not depend on IP3 signaling.

Intranasal Delivery of BACE1 siRNA and Rapamycin by Dual Targets Modified Nanoparticles for Alzheimer's Disease Therapy.

Alzheimer's disease (AD), as a progressive and irreversible brain disorder, remains the most universal neurodegenerative disease. No effective therapeutic methods are established yet due to the hindrance of the blood-brain barrier (BBB) and the complex pathological condition of AD.  Therefore, a multifunctional nanocarrier (Rapa@DAK/siRNA) for AD treatment is constructed to achieve small interfering RNA of ß-site precursor protein (APP) cleaving enzyme-1 (BACE1 siRNA) and rapamycin co-delivery into the brain, based on Aleuria aurantia lectin (AAL) and ß-amyploid (Aß)-binding peptides (KLVFF) modified PEGylated dendrigraft poly-l-lysines (DGLs) via intranasal administration. Nasal administration provides an effective way to deliver drugs directly into the brain through the nose-to-brain pathway. AAL, specifically binding to L-fucose located in the olfactory epithelium, endows Rapa@DAK/siRNA with high brain entry efficiency through intranasal administration. KLVFF peptide as an Aß targeting ligand and aggregation inhibitor enables nanoparticles to bind with Aß, inhibit Aß aggregation, and reduce toxicity. Meanwhile, the release of BACE1 siRNA and rapamycin is confirmed to reduce BACE1 expression, promote autophagy, and reduce Aß deposition. Rapa@DAK/siRNA is verified to improve the cognition of transgenic AD mice after intranasal administration. Collectively, the multifunctional nanocarrier provides an effective and potential intranasal avenue for combination therapy of AD.

Dapagliflozin as an autophagic enhancer via LKB1/AMPK/SIRT1 pathway in ovariectomized/D-galactose Alzheimer's rat model.

Autophagy and mitochondrial deficits are characteristics of early phase of Alzheimer's disease (AD). Sodium-glucose cotransporter-2 inhibitors have been nominated as a promising class against AD hallmarks. However, there are no available data yet to discuss the impact of gliflozins on autophagic pathways in AD. Peripherally, dapagliflozin's (DAPA) effect is mostly owed to autophagic signals. Thus, the goal of this study is to screen the power of DAPA centrally on LKB1/AMPK/SIRT1/mTOR signaling in the ovariectomized/D-galactose (OVX/D-Gal) rat model. Animals were arbitrarily distributed between 5 groups; the first group undergone sham operation, while remaining groups undergone OVX followed by D-Gal (150 mg/kg/day; i.p.) for 70 days. After 6 weeks, the third, fourth, and fifth groups received DAPA (1 mg/kg/day; p.o.); concomitantly with the AMPK inhibitor dorsomorphin (DORSO, 25 µg/rat, i.v.) in the fourth group and the SIRT1 inhibitor EX-527 (10 µg/rat, i.v.) in the fifth group. DAPA mitigated cognitive deficits of OVX/D-Gal rats, as mirrored in neurobehavioral task with hippocampal histopathological examination and immunohistochemical aggregates of p-Tau. The neuroprotective effect of DAPA was manifested by elevation of energy sensors; AMP/ATP ratio and LKB1/AMPK protein expressions along with autophagic markers; SIRT1, Beclin1, and LC3B expressions. Downstream the latter, DAPA boosted mTOR and mitochondrial function; TFAM, in contrary lessened BACE1. Herein, DORSO or EX-527 co-administration prohibited DAPA's actions where DORSO elucidated DAPA's direct effect on LKB1 while EX-527 mirrored its indirect effect on SIRT1. Therefore, DAPA implied its anti-AD effect, at least in part, via boosting hippocampal LKB1/AMPK/SIRT1/mTOR signaling in OVX/D-Gal rat model.

Long noncoding RNA BACE1-antisense transcript plays a critical role in Parkinson's disease via microRNA-214-3p/Cell death-inducing p53-target protein 1 axis.

This study aimed to analyze the function and latent mechanism of long noncoding RNA BACE1-antisense transcript (lncRNA BACE1-AS) in MPP+-induced SH-SY5Y cells. SH-SY5Y cells were cultivated in 1 mM MPP+ for 24 h to establish Parkinson's disease (PD) model in vitro. TargetScan and luciferase reporter assay were conducted to predict and verify the interaction between microRNA (miR)-214-3p and CDIP1 (Cell death-inducing p53-target protein 1). Cell viability, lactate dehydrogenase (LDH) release, and cell apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT), LDH, and flow cytometer. The secretion of inflammatory factors and representative biomarkers of oxidative stress, including reactive oxygen species (ROS) and superoxide dismutase (SOD) were assessed using enzyme-linked immunosorbent assay (ELISA) and specific assay kits. Results suggested that lncRNA BACE1-AS was over-expressed and miR-214-3p was under-expressed in MPP+-stimulated SH-SY5Y cells. Further analyses revealed that MPP+ inhibited cell viability; enhanced cell apoptosis, Cleaved Caspase-3 expression and Cleaved Caspase-3/GAPDH ratio; induced oxidative stress and inflammation in SH-SY5Y cells were inhibited by lncRNA BACE1-AS-siRNA transfection; and all these inhibitions were reversed by miR-214-3p inhibitor. In addition, we found that CDIP1 was directly targeted by miR-214-3p and up-regulated in MPP+-stimulated SH-SY5Y cells. Further functional assays suggested that CDIP1-plasmid reversed the effects of miR-214-3p mimic on MPP+-stimulated SH-SY5Y cells. In conclusion, lncRNA BACE1-AS regulates SH-SY5Y cell proliferation, apoptosis, inflammatory response, and oxidative stress through direct regulation of miR-214-3p/CDIP1 signaling axis, and could be a potential candidate associated with the diagnosis and treatment of PD.

Smad4 and γ-secretase knock-down effect on osteogenic differentiation mediated via Runx2 in canine mesenchymal stem cells.

Cell lineage determination during mesenchymal stem cell (MSCs) differentiation is a highly orchestrated process involving diverse signaling pathways and distinct classes of regulatory molecules. Bone morphogenetic protein (BMP) signaling positively influence the osteoblast lineage determination, whereas the Notch signaling may have a dimorphic action. Effective regenerative therapy for repairing bone defects requires ample knowledge of the signaling pathways responsible for the differentiation of MSCs. To elucidate the signaling pathways that drives canine bone-marrow derived MSCs towards osteogenic lineage, the current work was focused on BMP and Notch signaling. Target genes of Runx2, Smad4 and γ-secretase were silenced by short hairpin RNA (shRNA) in canine MSCs. Evaluation of the effect of gene silencing on in-vitro osteogenic differentiation potential was done by quantitative polymerase chain reaction (qPCR) for osteoblastic markers (Osteocalcin and Osteopontin) and Alizarin red S staining for the extracellular deposition of calcium. Silencing of Runx2 significantly reduced the osteocalcin and osteopontin gene expression while a similar trend was observed in the case of smad 4 silencing and their combination groups, but there was no difference found in Hey 1 expression. Runx2 and Smad4 silencing groups showed very less positive staining with Alizarin red S staining, whereas knockdown of γ-secretase and its combination groups showed reverse results as that of Runx2 and Smad4. Runx2 plays an indispensable part in directing the canine mesenchymal stem cells towards osteogenic lineage. Also, Smad-mediated BMP signaling induced the osteoblast-specific gene expression, whereas the notch pathway negatively regulated the osteogenic differentiation of canine MSCs.

ADAM10 attenuates the development of abdominal aortic aneurysms in a mouse model.

An abdominal aortic aneurysm (AAA) is a life­threatening disease associated with a high mortality rate. At present, surgery or minimally invasive interventions are used in clinical treatment, especially for small aneurysms. However, the benefits of surgical repair are not obvious, and AAA ruptures can be prevented by aneurysm therapy to inhibit the growth of small aneurysms. Therefore, evaluating effective drugs to treat small AAAs is urgently required. Chronic inflammation is the main pathological feature of aneurysmal tissues. The aim of the present study was to investigate the protective role and underlying mechanism of ADAM metallopeptidase domain 10 (ADAM10). In the present study, a mouse model of AAA was established via porcine pancreatic elastase perfusion for 5 min per day for 14 days. ADAM10 (6 mg/kg) was injected intraperitoneally following 3 days of porcine pancreatic elastase perfusion in the ADAM10 group and the treatment continued for 10 days. The maximum inner luminal diameters of the infrarenal abdominal aortas were measured using an animal ultrasound system. The levels of high mobility group box 1 (HMGB1) and soluble receptor for advanced glycosylation end products in serum samples were measured by ELISA. Hematoxylin and eosin and elastin van Gieson staining were performed to observe morphology, integrity of the elastin layers and elastin degradation. CD68 expression was detected by immunohistochemical staining. Reverse transcription­quantitative PCR and western blotting were used for detection of mRNA and protein levels. The gelatinolytic activities of MMP­2 and MMP­9 were quantified via gelatin zymography analysis. These results showed that ADAM10 inhibited HMGB1/RAGE/NF­κB signaling and MMP activity in the pathogenesis of pancreatic elastase­induced AAA, which provide insight into the molecular mechanism of AAA and suggested that ADAM10 may be a potential therapeutic target for AAA.

Attenuation of Pb-induced Aß generation and autophagic dysfunction via activation of SIRT1: Neuroprotective properties of resveratrol.

This study examined the neuroprotective properties of resveratrol (Res) and its target sirtuin1 (SIRT1) against lead (Pb)-mediated toxicity and discovered that both resveratrol treatment and SIRT1 overexpression restored blocked autophagic flux as well as reduced ß-amyloid (Aß) contents. Four-week-old male C57BL/6 mice were employed to consumed 0.2% Pb(Ac)2 solution or deionized water for 3 months followed by 12 months of Res (50 mg/kg BW) or vehicle gavage. In in vitro study, SH-SY5Y cells were pretreated with the SIRT1 activator SRT1720 (2 µM) or the inhibitor EX527 (2 µM) for 2 h, then 25 µM of Pb(Ac)2 was added and incubated for 48 h. Western blotting, RT-qPCR, enzyme-linked immunosorbent assay (ELISA), and Lyso-Tracker Red Staining were next used to estimate the potential alterations of the autophagic pathway as well as BACE1-mediated amyloid processing in response to Pb exposure, respectively. Our data revealed that Res treatment or SIRT1 activation resisted the induction of autophagy by Pb exposure through inhibition of LC3 and Beclin-1 expression and promoted the degradation of Aß and Tau phosphorylation. Besides, the SIRT1 activator (SRT1720) downregulated the expression of BACE1, the rate-limiting enzyme for Aß production, by inhibiting the activation of nuclear factor-κB (NF-κB) in Pb-treated SH-SY5Y cells, which resulted in reduced Aß production. Collectively, we verified the role of Res-SIRT1-autophagy as well as the SIRT1-NF-κB-BACE1 pathway in Pb-induced neuronal cell injury by in vivo or in vitro models. Our findings further elucidate the important role of SIRT1 and Res in counteracting Pb neurotoxicity, which may provide new interventions and targets for the subsequent treatment of neurodegenerative diseases.

Gamma Secretase Inhibitors in Cancer: A Current Perspective on Clinical Performance.

Gamma secretase inhibitors (GSIs), initially developed as Alzheimer's therapies, have been repurposed as anticancer agents given their inhibition of Notch receptor cleavage. The success of GSIs in preclinical models has been ascribed to induction of cancer stem-like cell differentiation and apoptosis, while also impairing epithelial-to-mesenchymal transition and sensitizing cells to traditional chemoradiotherapies. The promise of these agents has yet to be realized in the clinic, however, as GSIs have failed to demonstrate clinical benefit in most solid tumors with the notable exceptions of CNS malignancies and desmoid tumors. Disappointing clinical performance to date reflects important questions that remain to be answered. For example, what is the net impact of these agents on antitumor immune responses, and will they require concurrent targeting of tumor-intrinsic compensatory pathways? Addressing these limitations in our current understanding of GSI mechanisms will undoubtedly facilitate their rational incorporation into combinatorial strategies and provide a valuable tool with which to combat Notch-dependent cancers. In the present review, we provide a current understanding of GSI mechanisms, discuss clinical performance to date, and suggest areas for future investigation that might maximize the utility of these agents. IMPLICATIONS FOR PRACTICE: The performance of gamma secretase inhibitors (GSIs) in clinical trials generally has not reflected their encouraging performance in preclinical studies. This review provides a current perspective on the clinical performance of GSIs across various solid tumor types alongside putative mechanisms of antitumor activity. Through exploration of outstanding gaps in knowledge as well as reasons for success in certain cancer types, the authors identify areas for future investigation that will likely enable incorporation of GSIs into rational combinatorial strategies for superior tumor control and patient outcomes.

Confocal Raman microspectroscopic analysis on the time-dependent impact of DAPT, a γ-secretase inhibitor, to osteosarcoma cells.

Confocal Raman microspectroscopy (CRM) analysis provides subcellular compositional and morphology related information. In this study, we used CRM in conjunction with multivariate statistical analysis to elucidate the time-dependent impact of the γ-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) of osteosarcoma (OS) cells. The interactions of DAPT (20 µM) with a murine OS cell line K7M2 at 24 and 48 h were monitored. The spectral characteristics of drug action were identified to illustrate the cellular compositional alterations, showing that DAPT induced apoptosis by reducing the protein, lipid and nucleic acid content and structural changes. Multivariate algorithms, principal component analysis (PCA) and linear discriminant analysis (LDA) revealed a clear separation among cells in the untreated control (UT), 24H (DAPT-treated for 24 h), and 48H (DAPT-treated for 48 h) groups, achieving sensitivities of 100%, 96%, 100% and specificities of 98%, 100%, 100%, respectively. After point-scanned spectral imaging, K-means clustering analysis (KCA) was further used to visualize sub-cellular morphological changes and the underlying spectral characteristics in a temporal sequence. Compared with the UT group, Raman imaging results exhibited gradually increased nuclear division of OS cells with DAPT treatment duration extension, along with changes in the physiology of other organelles within the cell. By providing a unique perspective for understanding the temporary cellular responses to DAPT at molecular level, the achieved results form the foundation of strategies for the application of CRM and other Raman-based techniques for studying the therapeutic responses of other anticancer agents in cancer model systems.

Protective Effects of Compounds from Cimicifuga dahurica against Amyloid Beta Production in Vitro and Scopolamine-Induced Memory Impairment in Vivo.

Cimicifuga dahurica has traditionally been used as an antipyretic, analgesic, and anti-inflammatory agent and as a treatment for uterine and anal prolapse. This study has investigated the potential beneficial effects of this medicinal plant and its components on Alzheimer's disease (AD) with a focus on amyloid beta (Aß) production and scopolamine-induced memory impairment in mice. An ethanol extract from C. dahurica roots decreased Aß production in APP-CHO cells [Chinese hamster ovarian (CHO) cells stably expressing amyloid precursor protein (APP)], as determined by an enzyme-linked immunosorbent assay and Western blot analysis. Then, the compounds isolated from C. dahurica were tested for their antiamyloidogenic activities. Four compounds (1-4) efficiently interrupted Aß generation by suppressing the level of ß-secretase in APP-CHO cells. Moreover, the in vivo experimental results demonstrated that compound 4 improved the cognitive performances of mice with scopolamine-induced disruption on behavioral tests and the expression of memory-related proteins. Taken together, these results suggest that C. dahurica and its constituents are potential agents for preventing or alleviating the symptoms of AD.

NOTCH3 contributes to rhinovirus-induced goblet cell hyperplasia in COPD airway epithelial cells.

RATIONALE: Goblet cell hyperplasia (GCH) is one of the cardinal features of chronic obstructive pulmonary disease (COPD) and contributes to airways obstruction. Rhinovirus (RV), which causes acute exacerbations in patients with COPD, also causes prolonged airways obstruction. Previously, we showed that RV enhances mucin gene expression and increases goblet cell number in a COPD mouse model. This study examines whether RV causes sustained GCH in relevant models of COPD. METHODS: Mucociliary-differentiated COPD and normal airway epithelial cell cultures and mice with normal or COPD phenotype were infected with RV or sham and examined for GCH by immunofluorescence and/or mucin gene expression. In some experiments, RV-infected COPD cells and mice with COPD phenotype were treated with γ-secretase inhibitor or interleukin-13 neutralising antibody and assessed for GCH. To determine the contribution of NOTCH1/3 in RV-induced GCH, COPD cells transduced with NOTCH1/3 shRNA were used. RESULTS: RV-infected COPD, but not normal cell cultures, showed sustained GCH and increased mucin genes expression. Microarray analysis indicated increased expression of NOTCH1, NOTCH3 and HEY1 only in RV-infected COPD cells. Blocking NOTCH3, but not NOTCH1, attenuated RV-induced GCH in vitro. Inhibition of NOTCH signalling by γ-secretase inhibitor, but not neutralising antibody to IL-13, abrogated RV-induced GCH and mucin gene expression. CONCLUSIONS: RV induces sustained GCH via NOTCH3 particularly in COPD cells or mice with COPD phenotype. This may be one of the mechanisms that may contribute to RV-induced prolonged airways obstruction in COPD.

Berberine Alleviates Amyloid-Beta Pathology in the Brain of APP/PS1 Transgenic Mice via Inhibiting ß/γ-Secretases Activity and Enhancing α-Secretases.

BACKGROUND: Berberine (BBR) has neuroprotective effects on many brain diseases, including Alzheimer's disease (AD). Amyloid -beta (Aß) senile plaque is the most classical pathological hallmarks of AD. Aß produces from a sequential cleavage by ß-secretase (beta-site amyloid precursor protein cleaving enzyme 1, BACE1) and γ -secretase. The aim of our work was to investigate whether the neuroprotective effects of BBR on AD is related to inhibiting Aß pathology. METHOD: The cognitive function of mice was assessed by the Morris water maze (MWM) test. The Aß levels were determined by enzyme linked immunosorbent assay; the expression of APP, sAPPα, ADAM10 and ADAM17, sAPPß and BACE1 was detected by Western blotting; and the activity of γ -secretase complex (NCT, PS1, Aph-1α and Pen-2) was determined by Western blotting and immunohistochemistry. RESULTS: BBR improved learning and memory deficits of APP/PS1 mice. BBR decreased Aß levels in the hippocampus of APP/PS1 mice. BACE1 and sAPP -ß levels in the BBR-treated groups were significantly reduced in the hippocampus of AD mice. BBR markedly decreased the expression of PS1, Aph-1α and Pen-2, but had no effect on NCT. The levels of sAPPα, ADAM10 and ADAM17 in the hippocampus of BBR-treated mice significantly increased, compared with the control ones (P<0.05). CONCLUSION: BBR inhibits the activity of ß/γ-secretases, enhances α-secretases, and lowers the Aß level in the hippocampus of AD mice, and improves Alzheimer's-like cognitive impairment.

Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads.

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer's disease therapeutics.

Development of a central nervous system axonal myelination assay for high throughput screening.

BACKGROUND: Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. RESULTS: We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. CONCLUSIONS: We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.