Casein Kinase 2 dependent phosphorylation of eIF4B regulates BACE1 expression in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Increased Aβ production plays a fundamental role in the pathogenesis of the disease and BACE1, the protease that triggers the amyloidogenic processing of APP, is a key protein and a pharmacological target in AD. Changes in neuronal activity have been linked to BACE1 expression and Aβ generation, but the underlying mechanisms are still unclear. We provide clear evidence for the role of Casein Kinase 2 in the control of activity-driven BACE1 expression in cultured primary neurons, organotypic brain slices, and murine AD models. More specifically, we demonstrate that neuronal activity promotes Casein Kinase 2 dependent phosphorylation of the translation initiation factor eIF4B and this, in turn, controls BACE1 expression and APP processing. Finally, we show that eIF4B expression and phosphorylation are increased in the brain of APPPS1 and APP-KI mice, as well as in AD patients. Overall, we provide a definition of a mechanism linking brain activity with amyloid production and deposition, opening new perspectives from the therapeutic standpoint.Subject terms: Kinases, Alzheimer''s disease, Neuronal physiology, Pathogenesis     

Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling

The dimerisation of Raf kinases involves a central cluster within the kinase domain, the dimer interface (DIF). Yet, the importance of the DIF for the signalling potential of wild-type B-Raf (B-Raf(wt)) and its oncogenic counterparts remains unknown. Here, we show that the DIF plays a pivotal role for the activity of B-Raf(wt) and several of its gain-of-function (g-o-f) mutants. In contrast, the B-Raf(V600E), B-Raf(insT) and B-Raf(G469A) oncoproteins are remarkably resistant to mutations in the DIF. However, compared with B-Raf(wt), B-Raf(V600E) displays extended protomer contacts, increased homodimerisation and incorporation into larger protein complexes. In contrast, B-Raf(wt) and Raf-1(wt) mediated signalling triggered by oncogenic Ras as well as the paradoxical activation of Raf-1 by kinase-inactivated B-Raf require an intact DIF. Surprisingly, the B-Raf DIF is not required for dimerisation between Raf-1 and B-Raf, which was inactivated by the D594A mutation, sorafenib or PLX4720. This suggests that paradoxical MEK/ERK activation represents a two-step mechanism consisting of dimerisation and DIF-dependent transactivation. Our data further implicate the Raf DIF as a potential target against Ras-driven Raf-mediated (paradoxical) ERK activation.     

Impact of IL28B-related single nucleotide polymorphisms on liver histopathology in chronic hepatitis C genotype 2 and 3

Background and Aims

Recently, several genome-wide association studies have revealed that single nucleotide polymorphisms (SNPs) in proximity to IL28B predict spontaneous clearance of HCV infection as well as outcome following peginterferon and ribavirin therapy among HCV genotype 1 infected patients. The present study aimed to evaluate the impact of IL28B SNP variability on liver histology in the context of a phase III treatment trial (NORDynamIC) for treatment-naïve patients with chronic HCV genotype 2 or 3 infection, where pretreatment liver biopsies were mandatory.

Methods

Three hundred and thirty-nine Caucasian patients had samples available for IL28B genotyping (rs12979860) of whom 314 had pretreatment liver biopsies that were evaluated using the Ishak protocol, allowing for detailed grading and staging of liver histopathology.

Results

IL28B CC_rs12979860 genotype in HCV genotype 3 infected patients was associated with higher ALT levels (p<0.0001), higher AST to platelet ratio index (APRI; p = 0.001), and higher baseline viral load (p<0.0001) as compared to patients with the CT or TT genotypes. Additionally the CC_rs12979860 genotype entailed more pronounced portal inflammation (p = 0.02) and steatosis (p = 0.03). None of these associations were noted among HCV genotype 2 infected patients.

Conclusion

This study shows that the CC_rs12979860 SNP is associated with more pronounced liver histopathology in patients chronically infected with HCV genotype 3, which may be secondary to higher viral load. The finding that IL28B variability did not impact on liver pathology or viral load among genotype 2 infected patients implies that IL28B may differentially regulate the course of genotype 2 and 3 infection.     

Modified Nucleosides. II.1 Economical Synthesis of 2′,3′-Dideoxycytidine

Abstract

An economical two pot synthesis of 2′,3′-dideoxycytidine (2) from N⁴-acetyl-cytidine (4) has been developed. The key feature of this sequence is the in situ reductive elimination of a mixture of 1-(3-bromo-3-deoxy-2,5-di-O-acetyl-β-D-xylofuranosyl)-N⁴-acetylcytosine (5) and 1-(2-bromo-3-deoxy-3,5-di-O-acetyl-β-D-arabinofuranosyl)-N⁴-acetylcytosine (6) and subsequent hydrogenation of the resultant olefin over palladised charcoal.     

Monitoring gradient formation in a jet aerated bioreactor

Jet aerated loop reactors (JLRs) provide high mass transfer coefficients (k_La) and can be used for the intensification of mass transfer limited reactions. The jet loop reactor achieves higher k_La values than a stirred tank reactor (STR). The improvement relies on significantly higher local power inputs (～10⁴) than those obtainable with the STR. Operation at high local turnover rates requires efficient macromixing, otherwise reactor inhomogeneities might occur. If sufficient homogenization is not achieved, the selectivity of the reaction and the respective yields are decreased. Therefore, the balance between mixing and mass transfer in jet loop reactors is a critical design aspect. Monitoring the dissolved oxygen levels during the turnover of a steady sodium sulfite feed implied the abundance of gradients in the JLR. Prolonged mixing times at identical power input and aeration rates (～100%) were identified for the JLR in comparison to the STR. The insertion of a draft tube to the JLR led to a more homogenous dissolved oxygen distribution, but unfortunately a reduction of mixing time was not achieved. In case of increased medium viscosities as they may arise in high cell density cultivations, no gradient formation was detected. However, differences in medium viscosity significantly altered the mass transfer and mixing performance of the JLR.     

Impact of nozzle operation on mass transfer in jet aerated loop reactors. Characterization and comparison to an aerated stirred tank reactor

The impact of mass transfer on productivity can become a crucial aspect in the fermentative production of bulk chemicals. For highly aerobic bioprocesses the oxygen transfer rate (OTR) and productivity are coupled. The achievable space time yields can often be correlated to the mass transfer performance of the respective bioreactor. The oxygen mass transfer capability of a jet aerated loop reactor is discussed in terms of the volumetric oxygen mass transfer coefficient k_La [h^?1] and the energetic oxygen transfer efficiency E [kgO₂ kW^?1 h^?1]. The jet aerated loop reactor (JLR) is compared to the frequently deployed aerated stirred tank reactor. In jet aerated reactors high local power densities in the mixing zone allow higher mass transfer rates, compared to aerated stirred tank reactors. When both reactors are operated at identical volumetric power input and aeration rates, local k_La values up to 1.5 times higher are possible with the JLR. High dispersion efficiencies in the JLR can be maintained even if the nozzle is supplied with pressurized gas. For increased oxygen demands (above 120 mmol L^?1 h^?1) improved energetic oxygen transfer efficiencies of up to 100 % were found for a JLR compared to an aerated stirred tank reactor operating with Rushton turbines.