Neuronal Expression of Neural Nitric Oxide Synthase (nNOS) Protein is Suppressed by an Antisense RNA Transcribed from an NOS Pseudogene

Here, we show that a nitric oxide synthase (NOS) pseudogene is expressed in the CNS of the snail Lymnaea stagnalis. The pseudo-NOS transcript includes a region of significant antisense homology to a previously reported neuronal NOS (nNOS)-encoding mRNA. This suggested that the pseudo-NOS transcript acts as a natural antisense regulator of nNOS protein synthesis. In support of this, we show that both the nNOS-encoding and the pseudo-NOS transcripts are coexpressed in giant identified neurons (the cerebral giant cells) in the cerebral ganglion. Moreover, reverse transcription-PCR experiments on RNA isolated from the CNS establish that stable RNA-RNA duplex molecules do form between the two transcripts in vivo. Using an in vitro translation assay, we further demonstrate that the antisense region of the pseudogene transcript prevents the translation of nNOS protein from the nNOS-encoding mRNA. By analyzing NOS RNA and nNOS protein expression in two different identified neurons, we find that when both the nNOS-encoding and the pseudo-NOS transcripts are present in the same neuron, nNOS enzyme activity is substantially suppressed. Importantly, these results show that a natural antisense mechanism can mediate the translational control of nNOS expression in the Lymnaea CNS. Our findings also suggest that transcribed pseudogenes are not entirely without purpose and are a potential source of a new class of regulatory gene in the nervous system

Timed and targeted differential regulation of nitric oxide synthase (NOS) and anti-NOS genes by reward conditioning leading to long-term memory formation

In a number of neuronal models of learning, signaling by the neurotransmitter nitric oxide (NO), synthesized by the enzyme neuronal NO synthase (nNOS), is essential for the formation of long-term memory (LTM). Using the molluscan model system Lymnaea, we investigate here whether LTM formation is associated with specific changes in the activity of members of the NOS gene family: Lym-nNOS1, Lym-nNOS2, and the antisense RNA-producing pseudogene (anti-NOS). We show that expression of the Lym-nNOS1 gene is transiently upregulated in cerebral ganglia after conditioning. The activation of the gene is precisely timed and occurs at the end of a critical period during which NO is required for memory consolidation. Moreover, we demonstrate that this induction of the Lym-nNOS1 gene is targeted to an identified modulatory neuron called the cerebral giant cell (CGC). This neuron gates the conditioned feeding response and is an essential part of the neural network involved in LTM formation. We also show that the expression of the anti-NOS gene, which functions as a negative regulator of nNOS expression, is downregulated in the CGC by training at 4 h after conditioning, during the critical period of NO requirement. This appears to be the first report of the timed and targeted differential regulation of the activity of a group of related genes involved in the production of a neurotransmitter that is necessary for learning, measured in an identified neuron of known function. We also provide the first example of the behavioral regulation of a pseudogene

Suppression of nitric oxide (NO)-dependent behavior by double-stranded RNA-mediated silencing of a neuronal NO synthase gene

We have used double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to disrupt neuronal nitric oxide (NO) synthase (nNOS) gene function in the snail Lymnaea stagnalis and have detected a specific behavioral phenotype. The injection of whole animals with synthetic dsRNA molecules targeted to the nNOS-encoding mRNA reduces feeding behavior in vivo and fictive feeding in vitro and interferes with NO synthesis by the CNS. By showing that synthetic dsRNA targeted to the nNOS mRNA causes a significant and long-lasting reduction in the levels of Lym-nNOS mRNA, we verify that specific RNAi has occurred. Importantly, our results establish that the expression of nNOS gene is essential for normal feeding behavior. They also show that dsRNA can be used in the investigation of functional gene expression in the context of whole animal behavior, regardless of the availability of targeted mutation technologies

The temporal expression profile of a Nos3-related natural antisense RNA in the brain suggests a possible role in neurogenesis

Experimental work over the past several years has revealed an unexpected abundance of long natural antisense transcripts (NATs) in eukaryotic species. In light of the proposed role of such RNA molecules in the regulation of gene expression in the brain, attention is now focused on specific examples of neuronal NATs. Of particular interest are NATs that are complementary to mRNAs encoding nitric oxide synthase (NOS), the enzyme responsible for production of the important gaseous neurotransmitter nitric oxide (NO). Here we study the temporal expression profile of murine Nos3as NAT in the brain. Notably, Nos3as NAT is known to act as a negative regulator of Nos3 gene expression. The results of our quantitative analysis reveal differential expression of Nos3as NAT during embryonic and post-embryonic stages of development of the brain. Also, they show that the low levels of Nos3as NAT coincides with active neurogenesis. In addition we report on an inverse correlation between the relative expression level of Nos3as NAT and the level of Nos3 protein. Thus our data raise the hypothesis that the Nos3as NAT regulates neurogenesis through suppression of Nos3 gene activity. This idea is further supported by experiments conducted on the olfactory bulbs and cultured neuroblastoma cells

A CREB2-targeting microRNA is required for long-term memory after single-trial learning

Although single-trial induced long-term memories (LTM) have been of major interest in neuroscience, how LTM can form after a single episode of learning remains largely unknown. We hypothesized that the removal of molecular inhibitory constraints by microRNAs (miRNAs) plays an important role in this process. To test this hypothesis, first we constructed small non-coding RNA (sncRNA) cDNA libraries from the CNS of Lymnaea stagnalis subjected to a single conditioning trial. Then, by next generation sequencing of these libraries, we identified a specific pool of miRNAs regulated by training. Of these miRNAs, we focussed on Lym-miR-137 whose seed region shows perfect complementarity to a target sequence in the 3’ UTR of the mRNA for CREB2, a well-known memory repressor. We found that Lym-miR-137 was transiently up-regulated 1 h after single-trial conditioning, preceding a down-regulation of Lym-CREB2 mRNA. Furthermore, we discovered that Lym-miR-137 is co-expressed with Lym-CREB2 mRNA in an identified neuron with an established role in LTM. Finally, using an in vivo loss-of-function approach we demonstrated that Lym-miR-137 is required for single-trial induced LTM

Time dependent differential regulation of a novel long non-coding natural antisense RNA during long-term memory formation

Long natural antisense transcripts (NATs) have been demonstrated in significant numbers in a variety of eukaryotic organisms. They are particularly prevalent in the nervous system suggesting their importance in neural functions. However, the precise physiological roles of the overwhelming majority of long NATs remain unclear. Here we report on the characterization of a novel molluscan nitric oxide synthase (NOS)-related long non-coding NAT (Lym-NOS1AS). This NAT is spliced and polyadenylated and is transcribed from the non-template strand of the Lym-NOS1 gene. We demonstrate that the Lym-NOS1AS is co-expressed with the sense Lym-NOS1 mRNA in a key neuron of memory network. Also, we report that the Lym-NOS1AS is temporally and spatially regulated by one-trial conditioning leading to long term memory (LTM) formation. Specifically, in the cerebral, but not in the buccal ganglia, the temporal pattern of changes in Lym-NOS1AS expression after training correlates with the alteration of memory lapse and non-lapse periods. Our data suggest that the Lym-NOS1AS plays a role in the consolidation of nitric oxide-dependent LTM

ER residency of the ceramide phosphoethanolamine synthase SMSr relies on homotypic oligomerization mediated by its SAM domain

SMSr/SAMD8 is an ER-resident ceramide phosphoethanolamine synthase with a critical role in controlling ER ceramides and suppressing ceramide-induced apoptosis in cultured cells. SMSr-mediated ceramide homeostasis relies on the enzyme’s catalytic activity as well as on its N-terminal sterile α-motif or SAM domain. Here we report that SMSr-SAM is structurally and functionally related to the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane. Native gel electrophoresis indicates that both SAM domains form homotypic oligomers. Chemical crosslinking studies show that SMSr self-associates into ER-resident trimers and hexamers that resemble the helical oligomers formed by DGKδ-SAM. Residues critical for DGKδ-SAM oligomerization are conserved in SMSr-SAM and their substitution causes a dissociation of SMSr oligomers as well as a partial redistribution of the enzyme to the Golgi. Conversely, treatment of cells with curcumin, a drug disrupting ceramide and Ca2+ homeostasis in the ER, stabilizes SMSr oligomers and promotes retention of the enzyme in the ER. Our data provide first demonstration of a multi-pass membrane protein that undergoes homotypic oligomerization via its SAM domain and indicate that SAM-mediated self-assembly of SMSr is required for efficient retention of the enzyme in the ER

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes

Different concepts for creating antibacterial yet biocompatible surfaces: Adding bactericidal element, grafting therapeutic agent through COOH plasma polymer and their combination

Antibacterial coatings have become a rapidly developing field of research, strongly stimulated by the increasing urgency of identifying alternatives to the traditional administration of antibiotics. Such coatings can be deposited onto implants and other medical devices and prevent the inflammations caused by hospital-acquired infections. Nevertheless, the design of antibacterial yet biocompatible and bioactive surfaces is a challenge that biological community has faced for many years but the "materials of dream" have not yet been developed. In this work, the biocompatible yet antibacterial multi-layered films were prepared by a combination of magnetron sputtering (TiCaPCON film), ion implantation (Ag-doped TiCaPCON film), plasma polymerization (COOH layer), and the final immobilization of gentamicin (GM) and heparin (Hepa) molecules. The layer chemistry was thoroughly investigated by means of FTIR and X-ray photoelectron spectroscopies. It was found that the immobilization of therapeutic components occurs throughout the entire thickness of the plasma-deposited COOH layer. The influence of each type of bactericide (Ag+ ions, GM, and Hepa) on antibacterial activity and cell proliferation was analyzed. Our films were cytocompatible and demonstrated superior bactericidal efficiency toward antibioticresistant bacterial E. coli K261 strain. Increased toxicity while using the combination of Ag nanoparticles and COOH plasma polymer is discussed

Design, Performance, and Calibration of the CMS Hadron-Outer Calorimeter

The CMS hadron calorimeter is a sampling calorimeter with brass absorber and plastic scintillator tiles with wavelength shifting fibres for carrying the light to the readout device. The barrel hadron calorimeter is complemented with an outer calorimeter to ensure high energy shower containment in the calorimeter. Fabrication, testing and calibration of the outer hadron calorimeter are carried out keeping in mind its importance in the energy measurement of jets in view of linearity and resolution. It will provide a net improvement in missing \et measurements at LHC energies. The outer hadron calorimeter will also be used for the muon trigger in coincidence with other muon chambers in CMS