RNA-based translation activators for targeted gene upregulation.

Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present "translation-activating RNAs" (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs.

Trapping of Nicotinic Acetylcholine Receptor Ligands Assayed by Cellular Studies and PET Imaging.

A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membrane-permeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pK and high affinity for α4β2-type nicotinic receptors (α4β2Rs) are trapped in intracellular acidic vesicles containing α4β2Rs Nicotine, with lower pK and α4β2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligands in male and female mouse brain and identifying the trapping brain organelle as Golgi satellites (GSats).

Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome.

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity, and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that -glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these -glycosylation alterations, we discovered that they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite.

Development of fluorescence imaging probes for nicotinic acetylcholine α4β2 receptors.

Nicotinic acetylcholine α4β2 receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity α4β2 nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [F]nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging α4β2 nAChRs.

Selective and regulated trapping of nicotinic receptor weak base ligands and relevance to smoking cessation.

To better understand smoking cessation, we examined the actions of varenicline (Chantix) during long-term nicotine exposure. Varenicline reduced nicotine upregulation of α4β2-type nicotinic receptors (α4β2Rs) in live cells and neurons, but not for membrane preparations. Effects on upregulation depended on intracellular pH homeostasis and were not observed if acidic pH in intracellular compartments was neutralized.

Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitises locomotor responding to the drug.

Dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc) mediate the behavioral and motivational effects of many drugs of abuse, including nicotine. Repeated intermittent administration of these drugs, a pattern often associated with initial drug exposure, sensitises the reactivity of dopamine (DA) neurons in this pathway, enhances the locomotor behaviors the drugs emit, and promotes their pursuit and self-administration. Here we show that activation of nicotinic acetylcholine receptors (nAChRs) in the VTA, but not the NAcc, is essential for the induction of locomotor sensitisation by nicotine.

Nicotine-induced upregulation of native neuronal nicotinic receptors is caused by multiple mechanisms.

Nicotine causes changes in brain nicotinic acetylcholine receptors (nAChRs) during smoking that initiate addiction. Nicotine-induced upregulation is the long-lasting increase in nAChR radioligand binding sites in brain resulting from exposure. The mechanisms causing upregulation are not established.

Nicotine-induced upregulation of nicotinic receptors: underlying mechanisms and relevance to nicotine addiction.

A major hurdle in defining the molecular biology of nicotine addiction has been characterizing the different nicotinic acetylcholine receptor (nAChR) subtypes in the brain and how nicotine alters their function. Mounting evidence suggests that the addictive effects of nicotine, like other drugs of abuse, occur through interactions with its receptors in the mesolimbic dopamine system, particularly ventral tegmental area (VTA) neurons, where nicotinic receptors act to modulate the release of dopamine. The molecular identity of the nicotinic receptors responsible for drug seeking behavior, their cellular and subcellular location and the mechanisms by which these receptors initiate and maintain addiction are poorly defined.

Up-regulation of nicotinic receptors by nicotine varies with receptor subtype.

Recent evidence suggests that in addition to alpha4beta2 and alpha3-containing nicotinic receptors, alpha6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that alpha6beta2, like alpha3beta2 and alpha4beta2 receptors, formed high affinity epibatidine binding complexes that are pentameric, trafficked to the cell surface, and produced acetylcholine-evoked currents. Chronic nicotine exposure up-regulated alpha6beta2 receptors with differences in up-regulation time course and concentration dependence compared with alpha4beta2 receptors, the predominant high affinity nicotine binding site in brain.

Membrane associated estrogen receptors and related proteins: localization at the plasma membrane and the endoplasmic reticulum.

The female sex steroid, estradiol 17beta, mediates its effect through its association with estrogen receptor present in the target cell. So far the major emphasis has been given to the genomic actions of the hormone mediated by the nuclear estrogen receptors. Recent years have seen a shift in the ideas revealing the existence of estradiol binding entities both in the plasma membrane and the endoplasmic reticulum.

Proteins that mediate the nuclear entry of the goat uterine estrogen receptor activation factor (E-RAF): identification of a molecular basis for the inhibitory effect of progesterone on estrogen action.

A 66 kDa transport protein, tp66, has been identified as the protein that mediates the nuclear transport of the estrogen receptor activation factor (E-RAF). Indirect evidence shows that tp66 influences the transport of E-RAF mainly by recognizing the nuclear localization signals (NLS) on the latter. A 38 kDa nuclear pore complex protein (npcp38) has been identified to which tp66-E-RAF complex gets 'docked' prior to the nuclear entry of E-RAF.

Estradiol dependent anchoring of the goat uterine estrogen receptor activation factor (E-RAF) at the endoplasmic reticulum by a 55 kDa anchor protein (ap55).

The primary intracellular site of localization of the estrogen receptor activation factor (E-RAF) is shown here to be the endoplasmic reticulum where the protein remains anchored through an estrogen dependent mechanism. The retention of E-RAF by the endoplasmic reticulum is facilitated by two proteins: (1) a 55 kDa anchor protein (ap55) which is an integral membrane protein of the endoplasmic reticulum. ap55 is a high affinity estrogen binding protein.