Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in C. elegans

Mutations in pre-synaptic voltage gated calcium channels can lead to familial hemiplegic migraine type 1 (FHM1). While mammalian studies indicate that the migraine brain is hyperexcitable due to enhanced excitation or reduced inhibition, the molecular and cellular mechanisms underlying this excitatory/inhibitory (E/I) imbalance are poorly understood. We identified a gain-of-function (gf) mutation in the Caenorhabditis elegans CaV2 channel α1 subunit, UNC-2, which leads to increased calcium currents. unc-2(zf35gf) mutants exhibit hyperactivity and seizure-like motor behaviors. Expression of the unc-2 gene with FHM1 substitutions R192Q and S218L leads to hyperactivity similar to that of unc-2(zf35gf) mutants. unc-2(zf35gf) mutants display increased cholinergic-and decreased GABAergic-transmission. Moreover, increased cholinergic transmission in unc-2(zf35gf) mutants leads to an increase of cholinergic synapses and a TAX-6/calcineurin dependent reduction of GABA synapses. Our studies reveal mechanisms through which CaV2 gain-of-function mutations disrupt excitation-inhibition balance in the nervous system.Fil: Huang, Yung Chi. University of Massachussets; Estados UnidosFil: Pirri, Jennifer K.. University of Massachussets; Estados UnidosFil: Rayes, Diego Hernán. University of Massachussets; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Gao, Shangbang. Mount Sinai Hospital; Estados UnidosFil: Mulcahy, Ben. Mount Sinai Hospital; Estados UnidosFil: Grant, Jeff. University of Massachussets; Estados UnidosFil: Saheki, Yasunori. The Rockefeller University; Estados UnidosFil: Francis, Michael M.. University of Massachussets; Estados UnidosFil: Zhen, Mei. University of Toronto; Canadá. Mount Sinai Hospital; Estados UnidosFil: Alkema, Mark J.. University of Massachussets; Estados Unido

Citrin deficiency as a cause of chronic liver disorder mimicking non-alcoholic fatty liver disease

ArticleJOURNAL OF HEPATOLOGY. 49(5):810-820 (2008)journal articl

Left-right olfactory asymmetry results from antagonistic functions of voltage-activated calcium channels and the Raw repeat protein OLRN-1 in C. elegans

<p>Abstract</p> <p>Background</p> <p>The left and right AWC olfactory neurons in <it>Caenorhabditis elegans </it>differ in their functions and in their expression of chemosensory receptor genes; in each animal, one AWC randomly takes on one identity, designated AWC^OFF, and the contralateral AWC becomes AWC^ON. Signaling between AWC neurons induces left-right asymmetry through a gap junction network and a claudin-related protein, which inhibit a calcium-regulated MAP kinase pathway in the neuron that becomes AWC^ON.</p> <p>Results</p> <p>We show here that the asymmetry gene <it>olrn-1 </it>acts downstream of the gap junction and claudin genes to inhibit the calcium-MAP kinase pathway in AWC^ON. OLRN-1, a protein with potential membrane-association domains, is related to the <it>Drosophila </it>Raw protein, a negative regulator of JNK mitogen-activated protein (MAP) kinase signaling. <it>olrn-1 </it>opposes the action of two voltage-activated calcium channel homologs, <it>unc-2 </it>(CaV2) and <it>egl-19 </it>(CaV1), which act together to stimulate the calcium/calmodulin-dependent kinase CaMKII and the MAP kinase pathway. Calcium channel activity is essential in AWC^OFF, and the two AWC neurons coordinate left-right asymmetry using signals from the calcium channels and signals from <it>olrn-1</it>.</p> <p>Conclusion</p> <p><it>olrn-1 </it>and voltage-activated calcium channels are mediators and targets of AWC signaling that act at the transition between a multicellular signaling network and cell-autonomous execution of the decision. We suggest that the asymmetry decision in AWC results from the intercellular coupling of voltage-regulated channels, whose cross-regulation generates distinct calcium signals in the left and right AWC neurons. The interpretation of these signals by the kinase cascade initiates the sustained difference between the two cells.</p

Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening

To expand the available set of Baeyer–Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.

Proteomic analysis identifies proteins that continue to grow hepatic stem-like cells without differentiation

To understand the molecular mechanism underlying vigorous proliferative activity of hepatic stem-like (HSL) cells, we performed two-dimensional electrophoresis to identify the proteins statistically more abundant in rapidly growing undifferentiated HSL cells than in sodium butyrate-treated differentiated HSL cells. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Mascot search identified 6 proteins including prohibitin, vimentin, ezrin, annexin A3, acidic ribosomal phosphoprotein P0 and Grp75. Prohibitin and vimentin control the mitogen-activated protein (MAP) kinase pathway. Ezrin is phosphorylated by various protein-tyrosine kinases and modulates interactions between cytoskeletal and membrane proteins. Annexin A3 has a role in DNA synthesis. Acidic ribosomal phosphoprotein P0 and Grp75 play in protein synthesis. These results suggest that the proteins related to the MAP kinase cascade had some role in continuous proliferation of HSL cells without differentiation

Statin therapy inhibits remyelination in the central nervous system

Remyelination of lesions in the central nervous system contributes to neural repair following clinical relapses in multiple sclerosis. Remyelination is initiated by recruitment and differentiation of oligodendrocyte progenitor cells (OPCs) into myelinating oligodendrocytes. Simvastatin, a blood-brain barrier-permeable statin in multiple sclerosis clinical trials, has been shown to impact the in vitro processes that have been implicated in remyelination. Animals were fed a cuprizone-supplemented diet for 6 weeks to induce localized demyelination in the corpus callosum; subsequent return to normal diet for 3 weeks stimulated remyelination. Simvastatin was injected intraperitoneally during the period of coincident demyelination and OPC maturation (weeks 4 to 6), throughout the entire period of OPC responses (weeks 4 to 9), or during the remyelination-only phase (weeks 7 to 9). Simvastatin treatment (weeks 4 to 6) caused a decrease in myelin load and both Olig2(strong) and Nkx2.2(strong) OPC numbers. Simvastatin treatment (weeks 4 to 9 and 7 to 9) caused a decrease in myelin load, which was correlated with a reduction in Nkx2.2(strong) OPCs and an increase in Olig2(strong) cells, suggesting that OPCs were maintained in an immature state (Olig2(strong)/Nkx2.2(weak)). NogoA+ oligodendrocyte numbers were decreased during all simvastatin treatment regimens. Our findings suggest that simvastatin inhibits central nervous system remyelination by blocking progenitor differentiation, indicating the need to monitor effects of systemic immunotherapies that can access the central nervous system on brain tissue-repair processes

Identification of novel functional sequence variants in the gene for peptidase inhibitor 3

BACKGROUND: Peptidase inhibitor 3 (PI3) inhibits neutrophil elastase and proteinase-3, and has a potential role in skin and lung diseases as well as in cancer. Genome-wide expression profiling of chorioamniotic membranes revealed decreased expression of PI3 in women with preterm premature rupture of membranes. To elucidate the molecular mechanisms contributing to the decreased expression in amniotic membranes, the PI3 gene was searched for sequence variations and the functional significance of the identified promoter variants was studied. METHODS: Single nucleotide polymorphisms (SNPs) were identified by direct sequencing of PCR products spanning a region from 1,173 bp upstream to 1,266 bp downstream of the translation start site. Fourteen SNPs were genotyped from 112 and nine SNPs from 24 unrelated individuals. Putative transcription factor binding sites as detected by in silico search were verified by electrophoretic mobility shift assay (EMSA) using nuclear extract from Hela and amnion cell nuclear extract. Deviation from Hardy-Weinberg equilibrium (HWE) was tested by χ(2 )goodness-of-fit test. Haplotypes were estimated using expectation maximization (EM) algorithm. RESULTS: Twenty-three sequence variations were identified by direct sequencing of polymerase chain reaction (PCR) products covering 2,439 nt of the PI3 gene (-1,173 nt of promoter sequences and all three exons). Analysis of 112 unrelated individuals showed that 20 variants had minor allele frequencies (MAF) ranging from 0.02 to 0.46 representing "true polymorphisms", while three had MAF ≤ 0.01. Eleven variants were in the promoter region; several putative transcription factor binding sites were found at these sites by database searches. Differential binding of transcription factors was demonstrated at two polymorphic sites by electrophoretic mobility shift assays, both in amniotic and HeLa cell nuclear extracts. Differential binding of the transcription factor GATA1 at -689C>G site was confirmed by a supershift. CONCLUSION: The promoter sequences of PI3 have a high degree of variability. Functional promoter variants provide a possible mechanism for explaining the differences in PI3 mRNA expression levels in the chorioamniotic membranes, and are also likely to be useful in elucidating the role of PI3 in other diseases

Functional mapping of brain synapses by the enriching activity-marker SynaptoZip

Ideally, elucidating the role of specific brain circuits in animal behavior would require the ability to measure activity at all involved synapses, possibly with unrestricted field of view, thus even at those boutons deeply located into the brain. Here, we introduce and validate an efficient scheme reporting synaptic vesicle cycling in vivo. This is based on SynaptoZip, a genetically encoded molecule deploying in the vesicular lumen a bait moiety designed to capture upon exocytosis a labeled alien peptide, Synbond. The resulting signal is cumulative and stores the number of cycling events occurring at individual synapses. Since this functional signal is enduring and measurable both online and ex post, SynaptoZip provides a unique method for the analysis of the history of synaptic activity in regions several millimeters below the brain surface. We show its broad applicability by reporting stimulus-evoked and spontaneous circuit activity in wide cortical fields, in anesthetized and freely moving animals