Autoimmune RNA dysregulation and seizures: therapeutic prospects in neuropsychiatric lupus.

Lupus autoimmunity frequently presents with neuropsychiatric manifestations, but underlying etiology remains poorly understood. Human brain cytoplasmic 200 RNA (BC200 RNA) is a translational regulator in neuronal synapto-dendritic domains. Here, we show that a BC200 guanosine-adenosine dendritic transport motif is recognized by autoantibodies from a subset of neuropsychiatric lupus patients. These autoantibodies impact BC200 functionality by quasi irreversibly displacing two RNA transport factors from the guanosine-adenosine transport motif. Such anti-BC autoantibodies, which can gain access to brains of neuropsychiatric lupus patients, give rise to clinical manifestations including seizures. To establish causality, naive mice with a permeabilized blood-brain barrier were injected with anti-BC autoantibodies from lupus patients with seizures. Animals so injected developed seizure susceptibility with high mortality. Seizure activity was entirely precluded when animals were injected with lupus anti-BC autoantibodies together with BC200 decoy autoantigen. Seizures are a common clinical manifestation in neuropsychiatric lupus, and our work identifies anti-BC autoantibody activity as a mechanistic cause. The results demonstrate potential utility of BC200 decoys for autoantibody-specific therapeutic interventions in neuropsychiatric lupus.

Neuronal BC RNAs cooperate with eIF4B to mediate activity-dependent translational control.

In neurons, translational regulation of gene expression has been implicated in the activity-dependent management of synapto-dendritic protein repertoires. However, the fundamentals of stimulus-modulated translational control in neurons remain poorly understood. Here we describe a mechanism in which regulatory brain cytoplasmic (BC) RNAs cooperate with eukaryotic initiation factor 4B (eIF4B) to control translation in a manner that is responsive to neuronal activity. eIF4B is required for the translation of mRNAs with structured 5' untranslated regions (UTRs), exemplified here by neuronal protein kinase Mζ (PKMζ) mRNA. Upon neuronal stimulation, synapto-dendritic eIF4B is dephosphorylated at serine 406 in a rapid process that is mediated by protein phosphatase 2A. Such dephosphorylation causes a significant decrease in the binding affinity between eIF4B and BC RNA translational repressors, enabling the factor to engage the 40S small ribosomal subunit for translation initiation. BC RNA translational control, mediated via eIF4B phosphorylation status, couples neuronal activity to translational output, and thus provides a mechanistic basis for long-term plastic changes in nerve cells.

Delivery of RNA and its intracellular translation into protein mediated by SDS-CTAB vesicles: potential use in nanobiotechnology.

Catanionic vesicles are supramolecular aggregates spontaneously forming in water by electrostatic attraction between two surfactants mixed in nonstoichiometric ratios. The outer surface charges allow adsorption to the biomembrane by electrostatic interactions. The lipoplex thus obtained penetrates the cell by endocytosis or membrane fusion. We examined the possible cytotoxic effects and evaluated the transfection efficiency of one vesicle type as compared to known commercial carriers. We show that the individual components of two different vesicles types, CTAB (cetyltrimethylammonium bromide) and DDAB (didodecyldimethylammonium bromide) are detrimental for cell survival. We also assayed the cytotoxicity of SDS-DDAB vesicles and showed dose and time dependency, with the DDAB component being per se extremely cytotoxic. The transfection efficiency of exogenous RNA mediated by SDS-CTAB increases if vesicles assemble in the presence of the reporter RNA; finally, freezing abrogates the transfection ability. The results of our experimental strategy suggest that catanionic vesicles may be adopted in gene therapy and control of antiproliferative diseases.

Dual nature of translational control by regulatory BC RNAs.

In higher eukaryotes, increasing evidence suggests, gene expression is to a large degree controlled by RNA. Regulatory RNAs have been implicated in the management of neuronal function and plasticity in mammalian brains. However, much of the molecular-mechanistic framework that enables neuronal regulatory RNAs to control gene expression remains poorly understood. Here, we establish molecular mechanisms that underlie the regulatory capacity of neuronal BC RNAs in the translational control of gene expression. We report that regulatory BC RNAs employ a two-pronged approach in translational control. One of two distinct repression mechanisms is mediated by C-loop motifs in BC RNA 3' stem-loop domains. These C-loops bind to eIF4B and prevent the factor's interaction with 18S rRNA of the small ribosomal subunit. In the second mechanism, the central A-rich domains of BC RNAs target eIF4A, specifically inhibiting its RNA helicase activity. Thus, BC RNAs repress translation initiation in a bimodal mechanistic approach. As BC RNA functionality has evolved independently in rodent and primate lineages, our data suggest that BC RNA translational control was necessitated and implemented during mammalian phylogenetic development of complex neural systems.

Resveratrol exhibits a strong cytotoxic activity in cultured cells and has an antiviral action against polyomavirus: potential clinical use.

BACKGROUND: Resveratrol is a non flavonoid polyphenol compound present in many plants and fruits and, at especially high concentrations, in the grape berries of Vitis vinifera. This compound has a strong bioactivity and its cytoprotective action has been demonstrated, however at high concentrations the drug exhibits also an effective anti-proliferative action. We recently showed its ability to abolish the effects of oxidative stress in cultured cells. In this work we assayed the bioactivity of resveratrol as antiproliferative and antiviral drug in cultured fibroblasts. Studies by other Authors showed that this natural compound inhibits the proliferation of different viruses such as herpes simplex, varicella-zoster and influenza A. The results presented here show an evident toxic activity of the drug at high concentrations, on the other hand at sub-cytotoxic concentrations, resveratrol can effectively inhibit the synthesis of polyomavirus DNA. A possible interpretation is that, due to the damage caused by resveratrol to the plasma membrane, the transfer of the virus from the endoplasmic reticulum to the nucleus, may be hindered thus inhibiting the production of viral DNA. METHODS: The mouse fibroblast line 3T6 and the human tumor line HL60 were used throughout the work. Cell viability and vital cell count were assessed respectively, by the MTT assay and Trypan Blue staining. Cytotoxic properties and evaluation of viral DNA production by agarose gel electrophoresis were performed according to standard protocols. RESULTS: Our results show a clear dose dependent both cytotoxic and antiviral effect of resveratrol respectively at high and low concentrations. The cytotoxic action is exerted towards a stabilized cell-line (3T6) as well as a tumor-line (HL60). Furthermore the antiviral action is evident after the phase of virion entry, therefore data suggest that the drug acts during the synthesis of the viral progeny DNA. CONCLUSION: Resveratrol is cytotoxic and inhibits, in a dose dependent fashion, the synthesis of polyomavirus DNA in the infected cell. Furthermore, this inhibition is observed at non cytotoxic concentrations of the drug. Our data imply that cyto-toxicity may be attributed to the membrane damage caused by the drug and that the transfer of polyomavirus from the endoplasmic reticulum to the cytoplasm may be hindered. In conclusion, the cytotoxic and antiviral properties of resveratrol make it a potential candidate for the clinical control of proliferative as well as viral pathologies.

Alterations of the plasma membrane caused by murine polyomavirus proliferation: an electrorotation study.

In this report we investigate the alterations of the dielectric properties of the plasma membrane caused by the infection of cultured fibroblasts with murine polyomavirus. The approach consists in a well-established dielectric spectroscopy technique, electrorotation, which has been successfully used in our laboratory to study the alterations of the plasma membrane of cells exposed to various forms of stress. The response to viral proliferation was time dependent as shown by evaluation of the de novo synthesis of viral DNA. This response was paralleled by gradual damage of the membrane evidenced by alteration of the dielectric parameters, specific capacitance and conductance. The electrorotation results show a reduced effect on the dielectric properties of the membrane when infection is carried out in the presence of a natural oil (MEX). In this case a drastic reduction in viral DNA synthesis was also monitored, thus indicating an antiviral action of this product.

Differential cytotoxicity of MEX: a component of Neem oil whose action is exerted at the cell membrane level.

Neem oil is obtained from the seeds of the tree Azadirachta indica. Its chemical composition is very complex, being rich in terpenoids and limonoids, as well as volatile sulphur modified compounds. This work focused on the evaluation of a component of the whole Neem oil obtained by methanolic extraction and defined as MEX. Cytotoxicity was assessed on two different cell populations: a stabilized murine fibroblast line (3T6) and a tumor cell line (HeLa). The data presented here suggest a differential sensitivity of these two populations, the tumor line exhibiting a significantly higher sensitivity to MEX. The data strongly suggest that its toxic target is the cell membrane. In addition the results presented here imply that MEX may contain one or more agents that could find a potential use in anti-proliferative therapy.

Charge redistribution in adenosylribosyl transferase caused by substitution of a single amino acid residue.

Dielectric measurements in the frequency range 10(5)-10(8) Hz were performed on wild-type (wt) adenosylribosyl transferase and a mutant enzyme. The analysis of the dielectric relaxation curve allowed the estimation of the hydrodynamic radius and of the electric dipole moment. The first parameter remained unchanged in wt and mutant protein. The dipole moment of the mutant, however, was significantly increased. Implications on the electrostatic interactions between enzyme and substrate are discussed.