B cell-specific S1PR1 deficiency blocks prion dissemination between secondary lymphoid organs.

Many prion diseases are peripherally acquired (e.g., orally or via lesions to skin or mucous membranes). After peripheral exposure, prions replicate first upon follicular dendritic cells (FDC) in the draining lymphoid tissue before infecting the brain. However, after replication upon FDC within the draining lymphoid tissue, prions are subsequently propagated to most nondraining secondary lymphoid organs (SLO), including the spleen, by a previously underdetermined mechanism. The germinal centers in which FDC are situated produce a population of B cells that can recirculate between SLO. Therefore, we reasoned that B cells were ideal candidates by which prion dissemination between SLO may occur. Sphingosine 1-phosphate receptor (S1PR)1 stimulation controls the egress of T and B cells from SLO. S1PR1 signaling blockade sequesters lymphocytes within SLO, resulting in lymphopenia in the blood and lymph. We show that, in mice treated with the S1PR modulator FTY720 or with S1PR1 deficiency restricted to B cells, the dissemination of prions from the draining lymph node to nondraining SLO is blocked. These data suggest that B cells interacting with and acquiring surface proteins from FDC and recirculating between SLO via the blood and lymph mediate the initial propagation of prions from the draining lymphoid tissue to peripheral tissues.

Accelerated prion replication in, but prolonged survival times of, prion-infected CXCR3-/- mice.

Prion diseases have a significant inflammatory component. Glia activation, which is associated with increased production of cytokines and chemokines, may play an important role in disease development. Among the chemokines upregulated highly and early upregulated during scrapie infections are ligands of CXCR3. To gain more insight into the role of CXCR3 in a prion model, CXCR3-deficient (CXCR3(-/-)) mice were infected intracerebrally with scrapie strain 139A and characterized in comparison to similarly infected wild-type controls. CXCR3(-/-) mice showed significantly prolonged survival times of up to 30 days on average. Surprisingly, however, they displayed accelerated accumulation of misfolded proteinase K-resistant prion protein PrP(Sc) and 20 times higher infectious prion titers than wild-type mice at the asymptomatic stage of the disease, indicating that these PrP isoforms may not be critical determinants of survival times. As demonstrated by immunohistochemistry, Western blotting, and gene expression analysis, CXCR3-deficient animals develop an excessive astrocytosis. However, microglia activation is reduced. Quantitative analysis of gliosis-associated gene expression alterations demonstrated reduced mRNA levels for a number of proinflammatory factors in CXCR3(-/-) compared to wild-type mice, indicating a weaker inflammatory response in the knockout mice. Taken together, this murine prion model identifies CXCR3 as disease-modifying host factor and indicates that inflammatory glial responses may act in concert with PrP(Sc) in disease development. Moreover, the results indicate that targeting CXCR3 for treatment of prion infections could prolong survival times, but the results also raise the concern that impairment of microglial migration by ablation or inhibition of CXCR3 could result in increased accumulation of misfolded PrP(Sc).

A Method for Rapid Screening of Anilide-Containing AMPK Modulators Based on Computational Docking and Biological Validation.

Adenosine 5'-monophsphate-activated protein kinase (AMPK) is a crucial energy sensor for maintaining cellular homeostasis. Targeting AMPK may provide an alternative approach in treatment of various diseases like cancer, diabetes, and neurodegenerations. Accordingly, novel AMPK activators are frequently identified from natural products in recent years. However, most of such AMPK activators are interacting with AMPK in an indirect manner, which may cause off-target effects. Therefore, the search of novel direct AMPK modulators is inevitable and effective screening methods are needed. In this report, a rapid and straightforward method combining the use of in silico and in vitro techniques was established for selecting and categorizing huge amount of compounds from chemical library for targeting AMPK modulators. A new class of direct AMPK modulator have been discovered which are anilides or anilide-like compounds. In total 1,360,000 compounds were virtually screened and 17 compounds were selected after biological assays. Lipinski's rule of five assessment suggested that, 13 out of the 17 compounds are demonstrating optimal bioavailability. Proton acceptors constituting the structure of these compounds and hydrogen bonds with AMPK in the binding site appeared to be the important factors determining the efficacy of these compounds.

Mode of Action Analyses of Neferine, a Bisbenzylisoquinoline Alkaloid of Lotus (Nelumbo nucifera) against Multidrug-Resistant Tumor Cells.

Neferine, a bisbenzylisoquinoline alkaloid isolated from the green seed embryos of Lotus (Nelumbo nucifera Gaertn), has been previously shown to have various anti-cancer effects. In the present study, we evaluated the effect of neferine in terms of P-glycoprotein (P-gp) inhibition via in vitro cytotoxicity assays, R123 uptake assays in drug-resistant cancer cells, in silico molecular docking analysis on human P-gp and in silico absorption, distribution, metabolism, and excretion (ADME), quantitative structure activity relationships (QSAR) and toxicity analyses. Lipinski rule of five were mainly considered for the ADME evaluation and the preset descriptors including number of hydrogen bond donor, acceptor, hERG IC50, logp, logD were considered for the QSAR analyses. Neferine revealed higher toxicity toward paclitaxel- and doxorubicin-resistant breast, lung or colon cancer cells, implying collateral sensitivity of these cells toward neferine. Increased R123 uptake was observed in a comparable manner to the control P-gp inhibitor, verapamil. Molecular docking analyses revealed that neferine still interacts with P-gp, even if R123 was pre-bound. Bioinformatical ADME and toxicity analyses revealed that neferine possesses the druggability parameters with no predicted toxicity. In conclusion, neferine may allocate the P-gp drug-binding pocket and prevent R123 binding in agreement with P-gp inhibition experiments, where neferine increased R123 uptake.

The Calcium-Induced Regulation in the Molecular and Transcriptional Circuitry of Human Inflammatory Response and Autoimmunity.

Rheumatoid arthritis synovial fibroblasts (RASFs) are fundamental effector cells in RA driving the joint inflammation and deformities. Celastrol is a natural compound that exhibits a potent anti-arthritic effect promoting endoplasmic reticulum (ER) stress mediated by intracellular calcium (Ca2+) mobilization. Ca2+ is a second messenger regulating a variety of cellular processes. We hypothesized that the compound, celastrol, affecting cytosolic Ca2+ mobilization could serve as a novel strategy to combat RA. To address this issue, celastrol was used as a molecular tool to assay the inflammatory gene expression profile regulated by Ca2+. We confirmed that celastrol treatment mobilized cytosolic Ca2+ in patient-derived RASFs. It was found that 23 genes out of 370 were manipulated by Ca2+ mobilization using an inflammatory and autoimmunity PCR array following independent quantitative PCR validation. Most of the identified genes were downregulated and categorized into five groups corresponding to their cellular responses participating in RA pathogenesis. Accordingly, a signaling network map demonstrating the possible molecular circuitry connecting the functions of the products of these genes was generated based on literature review. In addition, a bioinformatics analysis revealed that celastrol-induced Ca2+ mobilization gene expression profile showed a novel mode of action compared with three FDA-approved rheumatic drugs (methotrexate, rituximab and tocilizumab). To the best of our knowledge, this is a pioneer work charting the Ca2+ signaling network on the regulation of RA-associated inflammatory gene expression.

N-Desmethyldauricine Induces Autophagic Cell Death in Apoptosis-Defective Cells via Ca2+ Mobilization.

Resistance of cancer cells to chemotherapy remains a significant problem in oncology. Mechanisms regulating programmed cell death, including apoptosis, autophagy or necrosis, in the treatment of cancers have been extensively investigated over the last few decades. Autophagy is now emerging as an important pathway in regulating cell death or survival in cancer therapy. Recent studies demonstrated variety of natural small-molecules could induce autophagic cell death in apoptosis-resistant cancer cells, therefore, discovery of novel autophagic enhancers from natural products could be a promising strategy for treatment of chemotherapy-resistant cancer. By computational virtual docking analysis, biochemical assays, and advanced live-cell imaging techniques, we have identified N-desmethyldauricine (LP-4), isolated from rhizoma of Menispermum dauricum DC as a novel inducer of autophagy. LP-4 was shown to induce autophagy via the Ulk-1-PERK and Ca2+/Calmodulin-dependent protein kinase kinase ß (CaMKKß)-AMPK-mTOR signaling cascades, via mobilizing calcium release through inhibition of SERCA, and importantly, lead to autophagic cell death in a panel of cancer cells, apoptosis-defective and apoptosis-resistant cells. Taken together, this study provides detailed insights into the cytotoxic mechanism of a novel autophagic compound that targeting the apoptosis resistant cancer cells, and new implication on drug discovery from natural products for drug resistant cancer therapy.

Evaluation of drugs for treatment of prion infections of the central nervous system.

Prion diseases are fatal and at present there are neither cures nor therapies available to delay disease onset or progression in humans. Inspired in part by therapeutic approaches in the fields of Alzheimer's disease and amyotrophic lateral sclerosis, we tested five different drugs, which are known to efficiently pass through the blood-brain barrier, in a murine prion model. Groups of intracerebrally prion-challenged mice were treated with the drugs curcumin, dapsone, ibuprofen, memantine and minocycline. Treatment with antibiotics dapsone and minocycline had no therapeutic benefit. Ibuprofen-treated mice showed severe adverse effects, which prevented assessment of therapeutic efficacy. Mice treated with low- but not high-dose curcumin and mice treated with memantine survived infections significantly longer than untreated controls (P<0.01). These results encourage further research efforts to improve the therapeutic effect of these drugs.

Role of galectin-3 in prion infections of the CNS.

Galectin-3 is a multi-functional protein and participates in mediating inflammatory reactions. The pronounced overexpression of galectin-3 in prion-infected brain tissue prompted us to study the role of this protein in a murine prion model. Immunofluorescence double-labelling identified microglia as the major cell type expressing galectin-3. Ablation of galectin-3 did not affect PrP(Sc)-deposition and development of gliosis. However, galectin-3(-/-)-mice showed prolonged survival times upon intracerebral and peripheral scrapie infections. Moreover, protein levels of the lysosomal activation marker LAMP-2 were markedly reduced in prion-infected galectin-3(-/-)-mice suggesting a role of galectin-3 in regulation of lysosomal functions. Lower mRNA levels of Beclin-1 and Atg5 in prion-infected wild-type and galectin-3(-/-)-mice indicated an impairment of autophagy although autophagosome formation was unchanged. The results point towards a detrimental role of galectin-3 in prion infections of the CNS and suggest that endo-/lysosomal dysfunction in combination with reduced autophagy may contribute to disease development.