An Inducer of VGF Protects Cells against ER Stress-Induced Cell Death and Prolongs Survival in the Mutant SOD1 Animal Models of Familial ALS

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease, and recent evidence has suggested that endoplasmic reticulum (ER) stress signaling is involved in the pathogenesis of ALS. Here we identified a small molecule, SUN N8075, which has a marked protective effect on ER stress-induced cell death, in an in vitro cell-based screening, and its protective mechanism was mediated by an induction of VGF nerve growth factor inducible (VGF): VGF knockdown with siRNA completely abolished the protective effect of SUN N8075 against ER-induced cell death, and overexpression of VGF inhibited ER-stress-induced cell death. VGF level was lower in the spinal cords of sporadic ALS patients than in the control patients. Furthermore, SUN N8075 slowed disease progression and prolonged survival in mutant SOD1 transgenic mouse and rat models of ALS, preventing the decrease of VGF expression in the spinal cords of ALS mice. These data suggest that VGF plays a critical role in motor neuron survival and may be a potential new therapeutic target for ALS, and SUN N8075 may become a potential therapeutic candidate for treatment of ALS

Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins

AbstractCyclosporine A (CsA) is an immunosuppressive drug that targets cyclophilins, cellular cofactors that regulate the immune system. Replication of hepatitis C virus (HCV) is suppressed by CsA, but the molecular basis of this suppression is still not fully understood. To investigate this suppression, we cultured HCV replicon cells (Con1, HCV genotype 1b, FLR-N cell) in the presence of CsA and obtained nine CsA-resistant FLR-N cell lines. We determined full-length HCV sequences for all nine clones, and chose two (clones #6 and #7) of the nine clones that have high replication activity in the presence of CsA for further analysis. Both clones showed two consensus mutations, one in NS3 (T1280V) and the other in NS5A (D2292E). Characterization of various mutants indicated that the D2292E mutation conferred resistance to high concentrations of CsA (up to 2μM). In addition, the missense mutation T1280V contributed to the recovery of colony formation activity. The effects of these mutations are also evident in two established HCV replicon cell lines—HCV-RMT ([1], genotype 1a) and JFH1 (genotype 2a). Moreover, three other missense mutations in NS5A—D2303H, S2362G, and E2414K—enhanced the resistance to CsA conferred by D2292E; these double or all quadruple mutants could resist approximately 8- to 25-fold higher concentrations of CsA than could wild-type Con1. These four mutations, either as single or combinations, also made Con1 strain resistant to two other cyclophilin inhibitors, N-methyl-4-isoleucine-cyclosporin (NIM811) or Debio-025. Interestingly, the changes in IC50 values that resulted from each of these mutations were the lowest in the Debio-025-treated cells, indicating its highest resistant activity against the adaptive mutation

Analysis of the mechanism by which BALB/c mice having prior immunization with nucleocapsid protein of SARS-CoV develop severe pneumonia after SARS-CoV infection

AbstractThe precise mechanism of severe acute respiratory syndrome (SARS), which is caused by SARS-associated coronavirus (SARS-CoV), is still unclear. We generated recombinant vaccinia virus (rVV) LC16m8 strain which simultaneously expresses four structural proteins of SARS-CoV, including nucleocapsid (N), membrane (M), envelop (E), spike (S) proteins (rVV-NMES) and reported that old BALB/c mice having prior immunization with rVV-NMES develop severe pneumonia similar to those of control mice though rVV-NMES-immunized mice showed lower pulmonary viral titer than in the control mice. Furthermore, we determined which SARS-CoV structural protein for the prior rVV-immunization is responsible for the severe pneumonia after the SARS-CoV infection as observed in the rVV-NMES-immunized mice. Old BALB/c mice were inoculated intradermally with rVV that expressed each structural proteins of SARS-CoV (rVV-N, -M, -E, or -S) with or without rVV-S and then infected intranasally with SARS-CoV more than 4 weeks later. At 9 days after SARS-CoV infection, the rVV-N-immunized mice show more severe pneumonia than in other groups. Furthermore, significant up-regulation of Th1 (IL-2)- and Th2 (IL-4 and IL-5)-bias cytokines and down-regulation of anti-inflammatory cytokine (IL-10 and TGF-β) were observed in rVV-N-immunized mice, resulting in the intensive infiltration of immunocompetent cells into the lung. In contrast, rVV-S-immunized mice showed only low pulmonary viral tier and slight pneumonia. However, the mice having co-immunization with both rVV-N and rVV-S showed severe pneumonia though their pulmonary viral titer was low. These results suggest that an excessive host immune response against the N protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS