Alternative sulfonylurea receptor expression defines metabolic sensitivity of K-ATP channels in dopaminergic midbrain neurons

ATP-sensitive potassium (K-ATP) channels couple the metabolic state to cellular excitability in various tissues. Several isoforms of the K-ATP channel subunits, the sulfonylurea receptor (SUR) and inwardly rectifying K channel (Kir6.X), have been cloned, but the molecular composition and functional diversity of native neuronal K-ATP channels remain unresolved. We combined functional analysis of K-ATP channels with expression profiling of K-ATP subunits at the level of single substantia nigra (SN) neurons in mouse brain slices using an RT-multiplex PCR protocol. In contrast to GABAergic neurons, single dopaminergic SN neurons displayed alternative co-expression of either SUR1, SUR2B or both SUR isoforms with Kir6.2. Dopaminergic SN neurons expressed alternative K-ATP channel species distinguished by significant differences in sulfonylurea affinity and metabolic sensitivity. In single dopaminergic SN neurons, co-expression of SUR1 + Kir6.2, but not of SUR2B + Kir6.2, correlated with functional K-ATP channels highly sensitive to metabolic inhibition. In contrast to wild-type, surviving dopaminergic SN neurons of homozygous weaver mouse exclusively expressed SUR1 + Kir6.2 during the active period of dopaminergic neurodegeneration. Therefore, alternative expression of K-ATP channel subunits defines the differential response to metabolic stress and constitutes a novel candidate mechanism for the differential vulnerability of dopaminergic neurons in response to respiratory chain dysfunction in Parkinson's disease.     

Three Novel Mutant Arylsulfatase A Alleles Causing Metachromatic Leukodystrophy

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. This leads to the accumulation of 3-O-sulfogalactosylceramide, which results in severe demyelination. Here we describe a novel non-sense mutation W124ter and two disease-causing missense mutations E382Q and C500F in arylsulfatase A gene. Another so far unknown allele harbors three sequence alterations: two polymorphisms (N350S, R496H) and a missense mutation (R288H). The R288H substitution and the N350S polymorphism have previously been found on one allele together with a polymorphism in a polyadenylation signal characteristic for the arylsulfatase A pseudodeficiency allele. The R496H has been shown to occur on another allele. The presence of the R288H, N350S, and R496H substitution on one allele in the absence of the polyadenylation site polymorphism shows that this allele has probably arisen by recombination between the nucleotides of codon 350 and 496.     

Novel factor Xa inhibitors based on a benzoic acid scaffold and incorporating a neutral P1 ligand

A series of novel, highly potent, achiral factor Xa inhibitors based on a benzoic acid scaffold and containing a chlorophenethyl moiety directed towards the protease S1 pocket is described. A number of structural features, such as the requirements of the P1, P4 and ester-binding pocket ligands were explored with respect to inhibition of factor Xa. Compound 46 was found to be the most potent compound in a series of antithrombotic secondary assays.     

Factor Xa inhibitors based on a 2-carboxyindole scaffold: SAR of neutral P1 substituents

A series of novel, highly potent 2-carboxyindole-based factor Xa inhibitors is described. Structural requirements for neutral ligands, which bind in the S1 pocket of factor Xa were investigated with the 2-carboxyindole scaffold. This privileged fragment assembly approach yielded a set of equipotent, selective inhibitors with structurally diverse neutral P1 substituents.     

Interleukin-18 enhances the production of interferon-gamma (IFN-gamma) by allergen-specific and unspecific stimulated cord blood mononuclear cells

BACKGROUND: IL-18 is a pleiotropic cytokine involved in the polarisation of T-cell response. This study was performed to determine whether or not IL-18 is detectable in phytohemagglutinin (PHA) or betalactoglobulin (BLG) stimulated supernatants of cord blood mononuclear cells (CBMC) and to study the in vitro effect of IL-18 on the interferon (IFN)-gaamma and IL-13 release of CBMC of healthy neonates. METHODS: CBMC of neonates were isolated by Ficoll density centrifugation. The cytokines IFN-gamma, IL-13 and IL-18 in the cell culture supernatants were measured using the ELISA technique following stimulation with a unspecific (PHA 20 microg/ml) and an allergen-specific stimulus (BLG 25 microg/ml). In order to study the in vitro effect of IL-18, CBMC were stimulated either with medium alone or with IL-18, IL-18 + PHA and IL-18 + BLG. RESULTS: IL-18 levels in supernatants of CBMC were low and did not vary significantly between unstimulated and PHA or BLG stimulated cell cultures (median 21.4; 23.5 and 15.5 pg/ml, respectively). IFN-gamma and IL-13 levels were significantly higher in response to PHA and BLG (PHA: IFN-gamma, 6154; IL-13, 4357; BLG: IFN-gamma, 801; IL-13, 249 pg/ml) compared to unstimulated cell cultures. The addition of IL-18 to PHA or BLG stimulated CBMC significantly enhanced the IFN-gamma release (PHA: 6154; PHA + IL-18: 13474, p = 0.0001; BLG: 801; BLG + IL-18: 1077, p = 0.008). In comparison to incubation without IL-18, the release of IL-13 was invariable or even reduced, when CBMC were stimulated with PHA + IL-18 (4026, p = 0.16) or BLG + IL-18 (124, p = 0.0001) compared to stimulation of CBMC with PHA (4357 pg/ml) or BLG (249 pg/ml) alone. CONCLUSIONS: IL-18 is detectable in supernatants of CBMC. We observed a significant effect of IL-18 + PHA as well as IL-18 + BLG on IFN-gamma release in vitro. Based on our findings we conclude that IL-18 could act as a strong TH1-inducing factor on stimulated CBMC also in vivo.     

Gene therapy: prospects for glycolipid storage diseases

Lysosomal storage diseases comprise a group of about 40 disorders, which in most cases are due to the deficiency of a lysosomal enzyme. Since lysosomal enzymes are involved in the degradation of various compounds, the diseases can be further subdivided according to which pathway is affected. Thus, enzyme deficiencies in the degradation pathway of glycosaminoglycans cause mucopolysaccharidosis, and deficiencies affecting glycopeptides cause glycoproteinosis. In glycolipid storage diseases enzymes are deficient that are involved in the degradation of sphingolipids. Mouse models are available for most of these diseases, and some of these mouse models have been used to study the applicability of in vivo gene therapy. We review the rationale for gene therapy in lysosomal disorders and present data, in particular, about trials in an animal model of metachromatic leukodystrophy. The data of these trials are compared with those obtained with animal models of other lysosomal diseases.     

ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization

Many point mutations in human Cu,Zn superoxide dismutase (SOD) cause familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder in heterozygotes. Here we show that these mutations cluster in protein regions influencing architectural integrity. Furthermore, crystal structures of SOD wild-type and FALS mutant H43R proteins uncover resulting local framework defects. Characterizations of beta-barrel (H43R) and dimer interface (A4V) FALS mutants reveal reduced stability and drastically increased aggregation propensity. Moreover, electron and atomic force microscopy indicate that these defects promote the formation of filamentous aggregates. The filaments resemble those seen in neurons of FALS patients and bind both Congo red and thioflavin T, suggesting the presence of amyloid-like, stacked beta-sheet interactions. These results support free-cysteine-independent aggregation of FALS mutant SOD as an integral part of FALS pathology. They furthermore provide a molecular basis for the single FALS disease phenotype resulting from mutations of diverse side-chains throughout the protein: many FALS mutations reduce structural integrity, lowering the energy barrier for fibrous aggregation.