The calcitonin receptor gene is a candidate for regulation of susceptibility to herpes simplex type 1 neuronal infection leading to encephalitis in rat.

Herpes simplex encephalitis (HSE) is a fatal infection of the central nervous system (CNS) predominantly caused by Herpes simplex virus type 1. Factors regulating the susceptibility to HSE are still largely unknown. To identify host gene(s) regulating HSE susceptibility we performed a genome-wide linkage scan in an intercross between the susceptible DA and the resistant PVG rat. We found one major quantitative trait locus (QTL), Hse1, on rat chromosome 4 (confidence interval 24.3-31 Mb; LOD score 29.5) governing disease susceptibility. Fine mapping of Hse1 using recombinants, haplotype mapping and sequencing, as well as expression analysis of all genes in the interval identified the calcitonin receptor gene (Calcr) as the main candidate, which also is supported by functional studies. Thus, using unbiased genetic approach variability in Calcr was identified as potentially critical for infection and viral spread to the CNS and subsequent HSE development.

Prion adsorption to stainless steel is promoted by nickel and molybdenum.

Prions are infectious agents resulting from the conversion of a normal cellular protein, PrP(C), to a misfolded species, PrP(Sc). Iatrogenic transmission of prions is known from surgical procedures involving stainless steel materials. Here, it was shown that stainless steel containing nickel and molybdenum binds PrP(Sc) more efficiently and transmits infection to cells in culture to a higher degree than if these elements are not present. Furthermore, both nickel and molybdenum alone adsorbed PrP(Sc), and nickel powder could be used to extract PrP(Sc) from dilute solutions, thus providing a simple approach to concentration of PrP(Sc). The fact that nickel and molybdenum in steel alloys increased the binding affinity, and bound infectivity, of PrP(Sc) is an important issue to consider in the manufacture of surgical instruments and abattoir tools.

Cannabinoids affect dendritic cell (DC) potassium channel function and modulate DC T cell stimulatory capacity.

Cannabinoids affect diverse biological processes, including functions of the immune system. With respect to the immune system, anti-inflammatory and immunosuppressive effects of cannabinoids have been reported. Cannabinoids stimulate G protein-coupled cannabinoid receptors CB1 and CB2. These receptors are found primarily on neurons. However, they are also found on dendritic cells (DC), which are recognized for their critical role in initiating and maintaining immune responses. Therefore, DC are potential targets for cannabinoids. We report in this study that cannabinoids reduced the DC surface expression of MHC class II molecules as well as their capacity to stimulate T cells. In the nervous system, CB1 receptor signaling modulates K(+) and Ca(2+) channels. Interestingly, cannabinoid-treated DC also showed altered voltage-gated potassium (K(V)) channel function. We speculate that attenuation of K(V) channel function via CB1 receptor signaling in DC may represent one mechanism by which cannabinoids alter DC function.

Inhibitors of the mitogen-activated protein kinase kinase 1/2 signaling pathway clear prion-infected cells from PrPSc.

Prions represent a unique class of infectious agents in which the normal cellular prion protein (PrPC) is converted to an abnormal isoform (PrPSc), which accumulates in the brain and constitutes the major, if not the only, component of the infectious particle. Factors that still remain to be identified may facilitate the conversion of PrPC to PrPSc. In the present study, we first demonstrated that a growth factor of the neurotrophin family, brain-derived neurotrophic factor (BDNF), stimulates the formation of PrPSc in a gonadotropin-releasing hormone-secreting neuronal cell line (GT1-1 cells) infected with the Rocky Mountain Laboratory (RML) strain of scrapie as determined by Western blot analysis. We then observed that the prion-infected cells can be cleared from PrPSc by treatment with three inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene and 2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one, as well as alpha-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl) benzeneacetonitrile, which passes the blood-brain barrier], a component of one of the intracellular signaling pathways activated by BDNF. The MEK1/2 inhibitors were also efficient in clearing PrPSc from prion-infected GT1-1 cells stimulated to accumulate high levels of PrPSc by enhanced serum concentrations in the medium or by the use of a serum-free neuron-specific neurobasal medium. PrPSc did not reappear in the cultures within 5 weeks after completion of treatment. We conclude that inhibitors of the MEK1/2 pathway can efficiently and probably irreversibly clear PrP(Sc) from prion-infected cells. The MEK pathway may therefore be a suitable target for therapeutic intervention in prion diseases.

Cathepsin B and L are involved in degradation of prions in GT1-1 neuronal cells.

In scrapie-infected cells, the abnormal isoform of the prion protein, PrP(Sc), accumulates in endosomes/lysosomes. In this study, the involvement of two lysosomal proteases, cathepsin B and L, in cellular processing of PrP(Sc) was analyzed in immortalized neuronal gonadotropin-releasing hormone cells (GT1-1) infected with scrapie. Treatment with inhibitors of either cathepsin B or L resulted in accumulation of PrP(Sc). Such an increased accumulation also occurred when the activities of both cathepsins were inhibited using RNA interference. We conclude that cathepsin B and L are involved in the degradation of PrP(Sc) in scrapie-infected GT1-1 cells and that they can compensate for each other's functions. This study shows that specific proteases, abundantly present in neurons, have the capacity to degrade PrP(Sc).

Scrapie protein degradation by cysteine proteases in CD11c+ dendritic cells and GT1-1 neuronal cells.

Dendritic cells (DC) of the CD11c(+) myeloid phenotype have been implicated in the spread of scrapie in the host. Previously, we have shown that CD11c(+) DC can cause a rapid degradation of proteinase K-resistant prion proteins (PrP(Sc)) in vitro, indicating a possible role of these cells in the clearance of PrP(Sc). To determine the mechanisms of PrP(Sc) degradation, CD11c(+) DC that had been exposed to PrP(Sc) derived from a neuronal cell line (GT1-1) infected with scrapie (ScGT1-1) were treated with a battery of protease inhibitors. Following treatment with the cysteine protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c), its ethyl ester (E-64d), and leupeptin, the degradation of PrP(Sc) was inhibited, while inhibitors of serine and aspartic and metalloproteases (aprotinin, pepstatin, and phosphoramidon) had no effect. An endogenous degradation of PrP(Sc) in ScGT1-1 cells was revealed by inhibiting the expression of cellular PrP (PrP(C)) by RNA interference, and this degradation could also be inhibited by the cysteine protease inhibitors. Our data show that PrP(Sc) is proteolytically cleaved preferentially by cysteine proteases in both CD11c(+) DC and ScGT1-1 cells and that the degradation of PrP(Sc) by proteases is different from that of PrP(C). Interference by protease inhibitors with DC-induced processing of PrP(Sc) has the potential to modify prion spread, clearance, and immunization in a host.

Processing and degradation of exogenous prion protein by CD11c(+) myeloid dendritic cells in vitro.

The immune system plays an important role in facilitating the spread of prion infections from the periphery to the central nervous system. CD11c(+) myeloid dendritic cells (DC) could, due to their subepithelial location and their migratory capacity, be early targets for prion infection and contribute to the spread of infection. In order to analyze mechanisms by which these cells may affect prion propagation, we studied in vitro the effect of exposing such DC to scrapie-infected GT1-1 cells, which produce the scrapie prion protein PrP(Sc). In this system, the DC efficiently engulfed the infected GT1-1 cells. Unexpectedly, PrP(Sc), which is generally resistant to protease digestion, was processed and rapidly degraded. Based on this observation we speculate that CD11c(+) DC may play a dual role in prion infections: on one hand they may facilitate neuroinvasion by transfer of the infectious agent as suggested from in vivo studies, but on the other hand they may protect against the infection by causing an efficient degradation of PrP(Sc). Thus, the migrating and highly proteolytic CD11c(+) myeloid DC may affect the balance between propagation and clearance of PrP(Sc) in the organism.