Chongsuk Ryou

Prions in skeletal muscle

Considerable evidence argues that consumption of beef products from cattle infected with bovine spongiform encephalopathy (BSE) prions causes new variant Creutzfeldt–Jakob disease. In an effort to prevent new variant Creutzfeldt–Jakob disease, certain “specified offals,” including neural and lymphatic tissues, thought to contain high titers of prions have been excluded from foods destined for human consumption [Phillips, N. A., Bridgeman, J. & Ferguson-Smith, M. (2000) in The BSE Inquiry (Stationery Office, London), Vol. 6, pp. 413–451]. Here we report that mouse skeletal muscle can propagate prions and accumulate substantial titers of these pathogens. We found both high prion titers and the disease-causing isoform of the prion protein (PrPSc) in the skeletal muscle of wild-type mice inoculated with either the Me7 or Rocky Mountain Laboratory strain of murine prions. Particular muscles accumulated distinct levels of PrPSc, with the highest levels observed in muscle from the hind limb. To determine whether prions are produced or merely accumulate intramuscularly, we established transgenic mice expressing either mouse or Syrian hamster PrP exclusively in muscle. Inoculating these mice intramuscularly with prions resulted in the formation of high titers of nascent prions in muscle. In contrast, inoculating mice in which PrP expression was targeted to hepatocytes resulted in low prion titers. Our data demonstrate that factors in addition to the amount of PrP expressed determine the tropism of prions for certain tissues. That some muscles are intrinsically capable of accumulating substantial titers of prions is of particular concern. Because significant dietary exposure to prions might occur through the consumption of meat, even if it is largely free of neural and lymphatic tissue, a comprehensive effort to map the distribution of prions in the muscle of infected livestock is needed. Furthermore, muscle may provide a readily biopsied tissue from which to diagnose prion disease in asymptomatic animals and even humans.

Functional Roles of Syk in Macrophage-Mediated Inflammatory Responses

Inflammation is a series of complex biological responses to protect the host from pathogen invasion. Chronic inflammation is considered a major cause of diseases, such as various types of inflammatory/autoimmune diseases and cancers. Spleen tyrosine kinase (Syk) was initially found to be highly expressed in hematopoietic cells and has been known to play crucial roles in adaptive immune responses. However, recent studies have reported that Syk is also involved in other biological functions, especially in innate immune responses. Although Syk has been extensively studied in adaptive immune responses, numerous studies have recently presented evidence that Syk has critical functions in macrophage-mediated inflammatory responses and is closely related to innate immune response. This review describes the characteristics of Syk-mediated signaling pathways, summarizes the recent findings supporting the crucial roles of Syk in macrophage-mediated inflammatory responses and diseases, and discusses Syk-targeted drug development for the therapy of inflammatory diseases.

Differential inhibition of prion propagation by enantiomers of quinacrine.

Prion diseases are fatal neurologic disorders caused by accumulation of a pathogenic isoform (PrPSc) of the prion protein (PrP). The recent discovery of the inhibitory action of quinacrine on PrPSc formation in scrapie-infected neuroblastoma (ScN2a) cells raised the possibility of a treatment for patients with prion disease. To investigate the efficacy of quinacrine enantiomers, we measured the inhibitory effect of these isomers on PrPSc formation in ScN2a cells. (S)-quinacrine exhibited superior antiprion activity compared with (R)-quinacrine and two generic quinacrines that appear to be racemates. Treatment with these various forms of quinacrine did not induce adverse changes affecting cell survival and the expression of marker proteins over a range of potentially therapeutic concentrations. Thus, quinacrine enantiomers demonstrated stereoselectivity on prion elimination but not cytotoxicity in ScN2a cells. Our results raise the possibility that in vivo treatment using one enantiomer of quinacrine may be superior to a racemic mixture, which is the form that is generally used when quinacrine is employed to treat parasitic diseases.

Cooperative binding of dominant-negative prion protein to kringle domains.

Conversion of the cellular prion protein (PrP(C)) to the pathogenic isoform (PrP(Sc)) is a major biochemical alteration in the progression of prion disease. This conversion process is thought to require interaction between PrP(C) and an as yet unidentified auxiliary factor, provisionally designated protein X. In searching for protein X, we screened a phage display cDNA expression library constructed from prion-infected neuroblastoma (ScN2a) cells and identified a kringle protein domain using full-length recombinant mouse PrP (recMoPrP(23-231), hereafter recMoPrP) expressing a dominant-negative mutation at codon 218 (recMoPrP(Q218K)). In vitro binding analysis using ELISA verified specific interaction of recMoPrP to kringle domains (K(1+2+3)) with higher binding by recMoPrP(Q218K) than by full-length recMoPrP without the mutation. This interaction was confirmed by competitive binding analysis, in which the addition of either a specific anti-kringle antibody or L-lysine abolished the interaction. Biochemical studies of the interactions between K(1+2+3) and various concentrations of both recMoPrP molecules demonstrated binding in a dose-dependent manner. A Hill plot analysis of the data indicates positive cooperative binding of both recMoPrP(Q218K) and recMoPrP to K(1+2+3) with stronger binding by recMoPrP(Q218K). Using full-length and an N-terminally truncated MoPrP(89-231), we demonstrate that N-terminal sequences enable PrP to bind strongly to K(1+2+3). Further characterization with truncated MoPrP(89-231) refolded in different conformations revealed that both alpha-helical and beta-sheet conformations bind to K(1+2+3). Our data demonstrate specific, high-affinity binding of a dominant-negative PrP as well as binding of other PrPs to K(1+2+3). The relevance of such interactions during prion pathogenesis remains to be established.

The inhibition of prions through blocking prion conversion by permanently charged branched polyamines of low cytotoxicity.

Branched polyamines are effective in inhibiting prions in a cationic surface charge density dependent manner. However, toxicity associated with branched polyamines, in general, often hampers the successful application of the compounds to treat prion diseases. Here, we report that constitutively maintained cationic properties in branched polyamines reduced the intrinsic toxicity of the compounds while retaining the anti-prion activities. In prion-infected neuroblastoma cells, quaternization of amines in polyethyleneimine (PEI) and polyamidoamine (PAMAM) dendrimers markedly increased the nontoxic concentration ranges of the compounds and still supported, albeit reduced, an appreciable level of anti-prion activity in clearing prions from the infected cells. Furthermore, quaternized PEI was able to degrade prions at acidic pH conditions and inhibit the in vitro prion propagation facilitated by conversion of the normal prion protein isoform to its misfolded counterpart, although such activities were decreased by quaternization. Quaternized PAMAM was least effective in degrading prions but efficiently inhibited prion conversion with the same efficacy as unmodified PAMAM. Our results suggest that quaternization represents an effective strategy for developing nontoxic branched polyamines with potent anti-prion activity. This study highlights the importance of polyamine structural control for developing polyamine-based anti-prion agents and understanding of an action mechanism of quaternized branched polyamines.

Pharmacokinetics of quinacrine in the treatment of prion disease.

BackgroundPrion diseases are caused by the accumulation of an aberrantly folded isoform of the prion protein, designated PrPSc. In a cell-based assay, quinacrine inhibits the conversion of normal host prion protein (PrPC) to PrPSc at a half-maximal concentration of 300 nM. While these data suggest that quinacrine may be beneficial in the treatment of prion disease, its penetration into brain tissue has not been extensively studied. If quinacrine penetrates brain tissue in concentrations exceeding that demonstrated for in vitro inhibition of PrPSc, it may be useful in the treatment of prion disease.MethodsOral quinacrine at doses of 37.5 mg/kg/D and 75 mg/kg/D was administered to mice for 4 consecutive weeks. Plasma and tissue (brain, liver, spleen) samples were taken over 8 weeks: 4 weeks with treatment, and 4 weeks after treatment ended.ResultsQuinacrine was demonstrated to penetrate rapidly into brain tissue, achieving concentrations up to 1500 ng/g, which is several-fold greater than that demonstrated to inhibit formation of PrPSc in cell culture. Particularly extensive distribution was observed in spleen (maximum of 100 μg/g) and liver (maximum of 400 μg/g) tissue.ConclusionsThe documented extensive brain tissue penetration is encouraging suggesting quinacrine might be useful in the treatment of prion disease. However, further clarification of the distribution of both intracellular and extracellular unbound quinacrine is needed. The relative importance of free quinacrine in these compartments upon the conversion of normal host prion protein (PrPC) to PrPSc will be critical toward its potential benefit.

Plasminogen stimulates propagation of protease-resistant prion protein in vitro

To clarify the role of plasminogen as a cofactor for prion propagation, we conducted functional assays using a cell-free prion protein (PrP) conversion assay termed protein misfolding cyclic amplification (PMCA) and prion-infected cell lines. Here, we report that plasminogen stimulates propagation of the protease-resistant scrapie PrP (PrP(Sc)). Compared to control PMCA conducted without plasminogen, addition of plasminogen in PMCA using wild-type brain material significantly increased PrP conversion, with an EC(50) = ∼56 nM. PrP conversion in PMCA was substantially less efficient with plasminogen-deficient brain material than with wild-type material. The activity stimulating PrP conversion was specific for plasminogen and conserved in its kringle domains. Such activity was abrogated by modification of plasminogen structure and interference of PrP-plasminogen interaction. Kinetic analysis of PrP(Sc) generation demonstrated that the presence of plasminogen in PMCA enhanced the PrP(Sc) production rate to ∼0.97 U/μl/h and reduced turnover time to ∼1 h compared to those (∼0.4 U/μl/h and ∼2.5 h) obtained without supplementation. Furthermore, as observed in PMCA, plasminogen and kringles promoted PrP(Sc) propagation in ScN2a and Elk 21(+) cells. Our results demonstrate that plasminogen functions in stimulating conversion processes and represents the first cellular protein cofactor that enhances the hypothetical mechanism of prion propagation.

Enhancement of protein misfolding cyclic amplification by using concentrated cellular prion protein source

Protein misfolding cyclic amplification (PMCA) is a cell-free assay mimicking the prion replication process. However, constraints affecting PMCA have not been well-defined. Although cellular prion protein (PrP(C)) is required for prion replication, the influence of PrP(C) abundance on PMCA has not been assessed. Here, we show that PMCA was enhanced by using mouse brain material in which PrP(C) was overexpressed. Tg(MoPrP)4112 mice overexpressing PrP(C) supported more sensitive and efficient PMCA than wild type mice. As brain homogenate of Tg(MoPrP)4112 mice was diluted with PrP(C)-deficient brain material, PMCA became less robust. Our studies suggest that abundance of PrP(C) is a determinant that directs enhancement of PMCA. PMCA established here will contribute to optimizing conditions to enhance PrP(Sc) amplification by using concentrated PrP(C) source and expands the use of this methodology.

Anti-inflammatory effect of methyl dehydrojasmonate (J2) is mediated by the NF-κB pathway

Inflammation as a major defense mechanism against pathogens is modulated by diverse microbial products. A variety of plant and microbial products interacting with Toll-like receptors initiate a wide spectrum of responses from phagocytosis to cytokine production, which modulates inflammation. Jasmonates are fatty acid-derived cyclopentanones produced by plants and lower eukaryotes that play an important role in the defense against insects. In this study, we are set up to define the molecular targets of J2 action. While the lipopolysaccharide (LPS) stimulation of macrophage cell line RAW264.7 induced TNF-α, IL-6, iNOS, and COX-2 that were associated with an increase in miR-155 and miR-146a, the J2 suppressed the induction of these inflammatory cytokines and enzymes as well as miR-155 in a dose-dependent manner. To assess the associations of miR-155 with inflammatory markers, we overexpressed miR-155 and found attenuation of COX-2 suppression with J2 treatment. Furthermore, J2 inhibited NF-κB, p65, and IκB but had no or only minimal effects on the mitogen-activated protein kinase pathway. In conclusion, the present study demonstrates that J2 suppresses LPS stimulation of RAW264.7 cells by targeting NF-κB pathways.

In Vitro Amplification of Misfolded Prion Protein Using Lysate of Cultured Cells

Protein misfolding cyclic amplification (PMCA) recapitulates the prion protein (PrP) conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrPC). PrPSc was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrPC in cell lysate was a critical factor to drive efficient PrPSc amplification, our results demonstrate that cell lysate in which PrPC is present abundantly serves as an excellent substrate source for PMCA.