Fritz Benseler

Direct Interaction of the Rat unc-13 Homologue Munc13-1 with the N Terminus of Syntaxin

unc-13 mutants in Caenorhabditis elegans are characterized by a severe deficit in neurotransmitter release. Their phenotype is similar to that of the C. elegans unc-18 mutation, which is thought to affect synaptic vesicle docking to the active zone. This suggests a crucial role for the unc-13 gene product in the mediation or regulation of synaptic vesicle exocytosis. Munc13-1 is one of three closely related rat homologues of unc-13. Based on the high degree of similarity between unc-13 and Munc13 proteins, it is thought that their essential function has been conserved from C. elegans to mammals. Munc13-1 is a brain-specific peripheral membrane protein with multiple regulatory domains that may mediate diacylglycerol, phospholipid, and calcium binding. In the present study, we demonstrate by three independent methods that the C terminus of Munc13-1 interacts directly with a putative coiled coil domain in the N-terminal part of syntaxin. Syntaxin is a component of the exocytotic synaptic core complex, a heterotrimeric protein complex with an essential role in transmitter release. Through this interaction, Munc13-1 binds to a subpopulation of the exocytotic core complex containing synaptobrevin, SNAP25 (synaptosomal-associated protein of 25 kDa), and syntaxin, but to no other tested syntaxin-interacting or core complex-interacting protein. The site of interaction in syntaxin is similar to the binding site for the unc-18 homologue Munc18, but different from that of all other known syntaxin interactors. These data indicate that unc-13-related proteins may indeed be involved in the mediation or regulation of synaptic vesicle exocytosis by modulating or regulating core complex formation. The similarity between the unc-13 and unc-18 phenotypes is paralleled by the coincidence of the binding sites for Munc13-1 and Munc18 in syntaxin. It is possible that the phenotype of unc-13 and unc-18 mutations is caused by the inability of the respective mutated gene products to bind to syntaxin.

A CAG repeat polymorphism of KCNN3 predicts SK3 channel function and cognitive performance in schizophrenia

KCNN3, encoding the small conductance calcium‐activated potassium channel SK3, harbours a polymorphic CAG repeat in the amino‐terminal coding region with yet unproven function. Hypothesizing that KCNN3 genotypes do not influence susceptibility to schizophrenia but modify its phenotype, we explored their contribution to specific schizophrenic symptoms. Using the Göttingen Research Association for Schizophrenia (GRAS) data collection of schizophrenic patients (n = 1074), we performed a phenotype‐based genetic association study (PGAS) of KCNN3. We show that long CAG repeats in the schizophrenic sample are specifically associated with better performance in higher cognitive tasks, comprising the capacity to discriminate, select and execute (p < 0.0001). Long repeats reduce SK3 channel function, as we demonstrate by patch‐clamping of transfected HEK293 cells. In contrast, modelling the opposite in mice, i.e. KCNN3 overexpression/channel hyperfunction, leads to selective deficits in higher brain functions comparable to those influenced by SK3 conductance in humans. To conclude, KCNN3 genotypes modify cognitive performance, shown here in a large sample of schizophrenic patients. Reduction of SK3 function may constitute a pharmacological target to improve cognition in schizophrenia and other conditions with cognitive impairment.

Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes

Fragile X syndrome (FXS) is mostly caused by a CGG triplet expansion in the fragile X mental retardation 1 gene (FMR1). Up to 60% of affected males fulfill criteria for autism spectrum disorder (ASD), making FXS the most frequent monogenetic cause of syndromic ASD. It is unknown, however, whether normal variants (independent of mutations) in the fragile X gene family (FMR1, FXR1, FXR2) and in FMR2 modulate autistic features. Here, we report an accumulation model of 8 SNPs in these genes, associated with autistic traits in a discovery sample of male patients with schizophrenia (N = 692) and three independent replicate samples: patients with schizophrenia (N = 626), patients with other psychiatric diagnoses (N = 111) and a general population sample (N = 2005). For first mechanistic insight, we contrasted microRNA expression in peripheral blood mononuclear cells of selected extreme group subjects with high‐ versus low‐risk constellation regarding the accumulation model. Thereby, the brain‐expressed miR‐181 species emerged as potential “umbrella regulator”, with several seed matches across the fragile X gene family and FMR2. To conclude, normal variation in these genes contributes to the continuum of autistic phenotypes.

Synthesis of Suitably-Protected Phosphoramidites of 2′-Fluoro-2′-Deoxyguanosine and 2′-Amino-2′-Deoxyguanosine for Incorporation Into Oligoribonucleotides

Abstract A novel synthesis of 2′-fluoro-2′-deoxyguanosine employing DAST as the fluorinating agent is presented. The preparation of its phosphoramidite as well as that of 2′-amino-2′-deoxyguanosine is also described.

A phenotype-based genetic association study reveals the contribution of neuregulin1 gene variants to age of onset and positive symptom severity in schizophrenia†‡

By pure endpoint diagnosis of the disease, the risk of developing schizophrenia has been repeatedly associated with specific variants of the neuregulin1 (NRG1) gene. However, the role of NRG1 in the etiology of schizophrenia has remained unclear. Since Nrg1 serves vital functions in early brain development of mice, we hypothesized that human NRG1 alleles codetermine developmentally influenced readouts of the disease: age of onset and positive symptom severity. We analyzed 1,071 comprehensively phenotyped schizophrenic/schizoaffective patients, diagnosed according to DSM‐IV‐TR, from the GRAS (Göttingen Research Association for Schizophrenia) Data Collection for genetic variability in the Icelandic region of risk in the NRG1 gene. For the case‐control analysis part of the study, we included 1,056 healthy individuals with comparable ethnicity. The phenotype‐based genetic association study (PGAS) was performed on the GRAS sample. Instead of a risk constellation, we detected that several haplotypic variants of NRG1 were, unexpectedly, less frequent in the schizophrenic than in the control sample (mean OR = 0.78, range between 0.68 and 0.85). In the PGAS we found that these “protective” NRG1 variants are specifically underrepresented in subgroups of schizophrenic subjects with early age of onset and high positive symptom load. The GRAS Data Collection as a prerequisite for PGAS has enabled us to associate protective NRG1 genotypes with later onset and milder course of schizophrenia.

1-Methylguanosine substitutions of the conserved guanosine residues inactive the hammerhead ribozyme

Abstract A study is described in which a methyl group is introduced to the N1-nitrogen of specific guanosine residues in the hammerhead ribozyme as a steric block to prevent hydrogen bonding interactions to the Watson-Crick face of each guanine base residue. The results of this study suggest the importance of hydrogen-bonding or other types of interactions to the Watson-Crick face of the three conserved G residues.

Synthesis of the oligodeoxyribonucleotide, d(CpTpGpGpApTpCpCpApG), and its substrate activity with the restriction endonuclease, BamHI

Abstract The synthesis of the DNA fragment, d(CpTpGpGpApTpCpCpApG), using a long-chain alkylamine-controlled pore glass bead polymer support, crystalline (9-phenyl-9-xanthenyl) nucleosides, and the bifunctional phosphorylating reagent, bis(1-benzotriazolyl)-2-chlorophenyl phosphate is described. For high coupling yields a mixed catalyst (1-methyl-imidazole + diisopropylethylamine) is required. The oligodeoxyribonucleotide shows activity with the DNA restriction endonuclease, Bam HI. Kinetic parameters have been determined for the reaction.