Dysbindin-1, a Schizophrenia-Related Protein, Functionally Interacts with the DNA- Dependent Protein Kinase Complex in an Isoform-Dependent Manner

DTNBP1 has been recognized as a schizophrenia susceptible gene, and its protein product, dysbindin-1, is down-regulated in the brains of schizophrenic patients. However, little is known about the physiological role of dysbindin-1 in the central nervous system. We hypothesized that disruption of dysbindin-1 with unidentified proteins could contribute to pathogenesis and the symptoms of schizophrenia. GST pull-down from human neuroblastoma lysates showed an association of dysbindin-1 with the DNA-dependent protein kinase (DNA-PK) complex. The DNA-PK complex interacts only with splice isoforms A and B, but not with C. We found that isoforms A and B localized in nucleus, where the kinase complex exist, whereas the isoform C was found exclusively in cytosol. Furthermore, results of phosphorylation assay suggest that the DNA-PK complex phosphorylated dysbindin-1 isoforms A and B in cells. These observations suggest that DNA-PK regulates the dysbindin-1 isoforms A and B by phosphorylation in nucleus. Isoform C does not contain exons from 1 to 6. Since schizophrenia-related single nucleotide polymorphisms (SNPs) occur in these introns between exon 1 and exon 6, we suggest that these SNPs might affect splicing of DTNBP1, which leads to impairment of the functional interaction between dysbindin-1 and DNA-PK in schizophrenic patients

Fragment of an endogenous inhibitor produced in Escherichia coli for calcium-activated neutral protease (CANP) retains an inhibitory activity

AbstractA C-terminal fragment of an endogenous rabbit liver inhibitor for calcium-activated neutral protease (CANP) was produced in Escherichia coli and its inhibitory activity was examined after purification. The truncated inhibitor (373 amino acid residues), which contains two internal repeat structures, inhibits 2 mol CANP whereas the native liver inhibitor (639 residues), containing four internal repeat structures, inhibits 4 mol CANP. This supports the hypothesis that the repeating unit is the functional unit of inhibition. The results also indicate that post-translational modification of the inhibitor is not essential for inhibition

BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin

Because mouse embryonic stem cells (mESCs) do not contribute to the formation of extraembryonic placenta when they are injected into blastocysts, it is believed that mESCs do not differentiate into trophoblast whereas human embryonic stem cells (hESCs) can express trophoblast markers when exposed to bone morphogenetic protein 4 (BMP4) in vitro. To test whether mESCs have the potential to differentiate into trophoblast, we assessed the effect of BMP4 on mESCs in a defined monolayer culture condition. The expression of trophoblast-specific transcription factors such as Cdx2, Dlx3, Esx1, Gata3, Hand1, Mash2, and Plx1 was specifically upregulated in the BMP4-treated differentiated cells, and these cells expressed trophoblast markers. These results suggest that BMP4 treatment in defined culture conditions enabled mESCs to differentiate into trophoblast. This differentiation was inhibited by serum or leukemia inhibitory factor, which are generally used for mESC culture. In addition, we studied the mechanism underlying BMP4-directed mESC differentiation into trophoblast. Our results showed that BMP4 activates the Smad pathway in mESCs inducing Cdx2 expression, which plays a crucial role in trophoblast differentiation, through the binding of Smad protein to the Cdx2 genomic enhancer sequence. Our findings imply that there is a common molecular mechanism underlying hESC and mESC differentiation into trophoblast

Alcoholism and Alternative Splicing of Candidate Genes

Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism

Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.Peer reviewe

Dopamine modulates acetylcholine release via octopamine and CREB signaling in Caenorhabditis elegans.

Animals change their behavior and metabolism in response to external stimuli. cAMP response element binding protein (CREB) is a signal-activated transcription factor that enables the coupling of extracellular signals and gene expression to induce adaptive changes. Biogenic amine neurotransmitters regulate CREB and such regulation is important for long-term changes in various nervous system functions, including learning and drug addiction. In Caenorhabditis elegans, the amine neurotransmitter octopamine activates a CREB homolog, CRH-1, in cholinergic SIA neurons, whereas dopamine suppresses CREB activation by inhibiting octopamine signaling in response to food stimuli. However, the physiological role of this activation is unknown. In this study, the effect of dopamine, octopamine, and CREB on acetylcholine signaling was analyzed using the acetylcholinesterase inhibitor aldicarb. Mutants with decreased dopamine signaling exhibited reduced acetylcholine signaling, and octopamine and CREB functioned downstream of dopamine in this regulation. This study demonstrates that the regulation of CREB by amine neurotransmitters modulates acetylcholine release from the neurons of C. elegans