Phosphodiesterase 2 inhibitors promote axonal outgrowth in organotypic slice co-cultures.

The development of appropriate models assessing the potential of substances for regeneration of neuronal circuits is of great importance. Here, we present procedures to analyze effects of substances on fiber outgrowth based on organotypic slice co-cultures of the nigrostriatal dopaminergic system in combination with biocytin tracing and tyrosine hydroxylase labeling and subsequent automated image quantification. Selected phosphodiesterase inhibitors (PDE-Is) were studied to identify their potential growth-promoting capacities. Immunohistochemical methods were used to visualize developing fibers in the border region between ventral tegmental area/substantia nigra co-cultivated with the striatum as well as the cellular expression of PDE2A and PDE10. The quantification shows a significant increase of fiber density in the border region induced by PDE2-Is (BAY60-7550; ND7001), comparable with the potential of the nerve growth factor and in contrast to PDE10-I (MP-10). Analysis of tyrosine hydroxylase-positive fibers indicated a significant increase after treatment with BAY60-7550 and nerve growth factor in relation to dimethyl sulfoxide. Additionally, a dose-dependent increase of intracellular cGMP levels in response to the applied PDE2-Is in PDE2-transfected HEK293 cells was found. In summary, our findings show that PDE2-Is are able to significantly promote axonal outgrowth in organotypic slice co-cultures, which are a suitable model to assess growth-related effects in neuro(re)generation.

Crystal structure of the interleukin-4/receptor alpha chain complex reveals a mosaic binding interface.

Interleukin-4 (IL-4) is a principal regulatory cytokine during an immune response and a crucial determinant for allergy and asthma. IL-4 binds with high affinity and specificity to the ectodomain of the IL-4 receptor alpha chain (IL4-BP). Subsequently, this intermediate complex recruits the common gamma chain (gamma c), thereby initiating transmembrane signaling. The crystal structure of the intermediate complex between human IL-4 and IL4-BP was determined at 2.3 A resolution. It reveals a novel spatial orientation of the two proteins, a small but unexpected conformational change in the receptor-bound IL-4, and an interface with three separate clusters of trans-interacting residues. Novel insights on ligand binding in the cytokine receptor family and a paradigm for receptors of IL-2, IL-7, IL-9, and IL-15, which all utilize gamma c, are provided.

Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain.

The specific high-affinity binding of interleukin-4 (IL-4) to its receptor alpha chain is the crucial primary event during IL-4 signalling. Single crystals, suitable for high resolution diffraction studies, have been obtained from a complex between IL-4 and the ectodomain of the receptor alpha chain, also called IL-4-binding protein (IL-4BP). The orthorhombic crystals are in spacegroup P2(1)2(1)2(1) with cell constants a = 5.038 nm, b = 6.841 nm, c = 10.95 nm and diffract to a resolution of at least 0.25 nm when exposed to synchrotron radiation. The volume of the unit cell suggests the presence of a 1:1 IL-4/IL-4BP complex and HPLC analysis of the crystals confirmed that IL-4 and IL-4BP were present in equimolar amounts. An IL-4 variant comprising a total of four methionine residues was generated, labelled with selenomethionine and crystallised in complex with IL-4BP. The crystals are isomorphous to that of the complex with normal IL-4 and therefore can be used to solve the crystallographic phase problem by the method of multiple anomalous diffraction (MAD). The crystal structure of the IL-4/IL-4BP complex will help to understand how IL-4 and other helical cytokines bind and activate their cognate receptor.

Global and local determinants for the kinetics of interleukin-4/interleukin-4 receptor alpha chain interaction. A biosensor study employing recombinant interleukin-4-binding protein.

An engineered interleukin-4-binding protein (IL4-BP) representing the extracellular domain of the human interleukin-4 (IL-4) receptor alpha chain was expressed in Sf9 cells. The purified IL4-BP was immobilized via a single biotinylated SH group near the carboxyl end to a biosensor matrix and analysed in real time for interaction with IL-4 and IL-4 variants. IL-4 was bound to IL4-BP at a molar ratio of approximately 1:1. The association and dissociation at pH 7.4 and 150 mM NaCl had rate constants of 1.9 +/- 0.3 x 10(7) M-1 s-1 and 2 +/- 1 x 10(-3) s-1, respectively. Glycosylation and engineered amino acid substitutions of IL4-BP did not alter the kinetic constants as shown by a parallel analysis of IL4-BP variants produced in Escherichia coli or Chinese hamster ovary cells. The rate of association was only slightly affected in binding-deficient variants [E9Q]IL-4 and [R88Q]IL-4 and by acidic pH down to values of 4.5, but it was reduced up to fivefold at higher ionic strength. The rate of dissociation was increased 70-fold and 150-fold with the IL-4 variants and fivefold at an acidic pH of 4.5, but it was not affected by high ionic strength. Temperatures between 6 degrees C and 37 degrees C yielded similar rates of IL-4 dissociation and only a marginally reduced rate of IL-4 association at 6 degrees C. These results indicate that the high-affinity binding of IL-4 to its receptor (Kd approximately 100 pM) is mainly the result of an unusually high association rate. The IL-4/IL4-BP interaction appears to be dominated by charge effects. The exceedingly high rate of IL-4/IL4-BP association is augmented by the overall electrostatic potentials of both proteins (electrostatic steering). Localized charges and the formation of ion pairs may control the rate of complex dissociation.