ERß agonist alters RNA splicing factor expression and has a longer window of antidepressant effectiveness than estradiol after long-term ovariectomy

Background: Estrogen therapy (ET), an effective treatment for perimenopausal depression, often fails to ameliorate symptoms when initiated late after the onset of menopause. Our previous work has suggested that alternative splicing of RNA might mediate these differential effects of ET. Methods: Female Sprague­Dawley rats were treated with estradiol (E2) or vehicle 6 days (early ET) or 180 days (late ET) after ovariectomy (OVX). We investigated the differential expression of RNA splicing factors and tryptophan hydroxylase 2 (TPH2) protein using a customized RT2 Profiler PCR Array, reverse-transcription polymerase chain reaction, immunoprecipitation and behaviour changes in clinically relevant early and late ET. Results: Early ET, but not late ET, prolonged swimming time in the forced swim test and reduced anxiety-like behaviours in the elevated plus maze. It reversed OVX-increased (SFRS7 and SFRS16) or OVX-decreased (ZRSR2 and CTNNB1) mRNA levels of splicing factors and ERß splicing changes in the brains of OVX rats. Early ET, but not late ET, also increased the expression of TPH2 and decreased monoamine oxidase A levels in the dorsal raphe in the brains of OVX rats. In late ET, only diarylpropionitrile (an ERß-specific agonist) achieved similar results ­ not E2 (an ERα and ERß agonist) or propylpyrazoletriol (an ERα-specific agonist). Limitations: Our experimental paradigm mimicked early and late ET in the clinical setting, but the contribution of age and OVX might be difficult to distinguish. Conclusion: These findings suggest that ERß alternative splicing and altered responses in the regulatory system for serotonin may mediate the antidepressant efficacy of ET associated with the timing of therapy initiation. It is likely that ERß-specific ligands would be effective estrogen-based antidepressants late after the onset of menopause.

Cleavage and Structural Transitions during Maturation of Staphylococcus aureus Bacteriophage 80α and SaPI1 Capsids.

In the tailed bacteriophages, DNA is packaged into spherical procapsids, leading to expansion into angular, thin-walled mature capsids. In many cases, this maturation is accompanied by cleavage of the major capsid protein (CP) and other capsid-associated proteins, including the scaffolding protein (SP) that serves as a chaperone for the assembly process. Staphylococcus aureus bacteriophage 80α is capable of high frequency mobilization of mobile genetic elements called S. aureus pathogenicity islands (SaPIs), such as SaPI1. SaPI1 redirects the assembly pathway of 80α to form capsids that are smaller than those normally made by the phage alone. Both CP and SP of 80α are N-terminally processed by a host-encoded protease, Prp. We have analyzed phage mutants that express pre-cleaved or uncleavable versions of CP or SP, and show that the N-terminal sequence in SP is absolutely required for assembly, but does not need to be cleaved in order to produce viable capsids. Mutants with pre-cleaved or uncleavable CP display normal viability. We have used cryo-EM to solve the structures of mature capsids from an 80α mutant expressing uncleavable CP, and from wildtype SaPI1. Comparisons with structures of 80α and SaPI1 procapsids show that capsid maturation involves major conformational changes in CP, consistent with a release of the CP N-arm by SP. The hexamers reorganize during maturation to accommodate the different environments in the 80α and SaPI1 capsids.

Derepression of SaPIbov1 Is Independent of φNM1 Type 2 dUTPase Activity and Is Inhibited by dUTP and dUMP.

Staphylococcus aureus is an opportunistic human pathogen able to transfer virulence genes to other cells through the mobilization of S. aureus pathogenicity islands (SaPIs). SaPIs are derepressed and packaged into phage-like transducing particles by helper phages like 80α or φNM1. Phages 80α and φNM1 encode structurally distinct dUTPases, Dut80α (type 1) and DutNM1 (type 2). Both dUTPases can interact with the SaPIbov1 Stl master repressor, leading to derepression and mobilization. That two structurally distinct dUTPases bind the same repressor led us to speculate that dUTPase activity may be important to the derepression process. In type 1 dUTPases, Stl binding is inhibited by dUTP. The purpose of this study was to assess the involvement of dUTP binding and dUTPase activity in derepression by DutNM1. DutNM1 activity mutants were created and tested for dUTPase activity using a novel NMR-based assay. We found that all DutNM1 null activity mutants interacted with the SaPIbov1 Stl C-terminal domain, formed DutNM1-Stl heterodimers, and caused the release of the Pstr promoter. However, promoter release was inhibited in the presence of dUTP or dUMP. We tested two φNM1 mutant phages that had null enzyme activity and found that they could still mobilize SaPIbov1. These results show that only the apo form of DutNM1 is active in Stl derepression and that dUTPase activity is not necessary for the mobilization of SaPIbov1 by DutNM1.

The Type 2 dUTPase of Bacteriophage ϕNM1 Initiates Mobilization of Staphylococcus aureus Bovine Pathogenicity Island 1.

Staphylococcus aureus pathogenicity islands (SaPIs) are genetic elements that are mobilized by specific helper phages. The initial step in mobilization is the derepression of the SaPI by the interaction of a phage protein with the SaPI master repressor Stl. Stl proteins are highly divergent between different SaPIs and respond to different phage-encoded derepressors. One such SaPI, SaPIbov1, is derepressed by the dUTPase (Dut) of bacteriophage 80α (Dut80α) and its phage ϕ11 homolog, Dut11. We previously showed that SaPIbov1 could also be mobilized by phage ϕNM1, even though its dut gene is not homologous with that of 80α. Here, we show that ϕNM1 dut encodes a type 2 dUTPase (DutNM1), which has an α-helical structure that is distinct from the type 1 trimeric, ß-sheet structure of Dut80α. Deletion of dutNM1 abolishes the ability of ϕNM1 to mobilize SaPIbov1. Like Dut80α, DutNM1 forms a direct interaction with SaPIbov1 Stl both in vivo and in vitro, leading to inhibition of the dUTPase activity and Stl release from its target DNA. This work provides novel insights into the diverse mechanisms of genetic mobilization in S. aureus.

Allopregnanolone reinstates tyrosine hydroxylase immunoreactive neurons and motor performance in an MPTP-lesioned mouse model of Parkinson's disease.

Restorative/protective therapies to restore dopamine neurons in the substantia nigra pars compacta (SNpc) are greatly needed to effectively change the debilitating course of Parkinson's disease. In this study, we tested the therapeutic potential of a neurogenic neurosteroid, allopregnanolone, in the restoration of the components of the nigrostriatal pathway in MPTP-lesioned mice by measuring striatal dopamine levels, total and tyrosine hydroxylase immunoreactive neuron numbers and BrdU-positive cells in the SNpc. An acute treatment (once/week for two weeks) with allopregnanolone restored the number of tyrosine hydroxylase-positive and total cell numbers in the SNpc of MPTP-lesioned mice, even though this did not increase striatal dopamine. It was also noted that MPTP treated mice to which allopregnanolone was administered had an increase in BrdU-positive cells in the SNpc. The effects of allopregnanolone in MPTP-lesioned mice were more apparent in mice that underwent behavioral tests. Interestingly, mice treated with allopregnanolone after MPTP lesion were able to perform at levels similar to that of non-lesioned control mice in a rotarod test. These data demonstrate that allopregnanolone promotes the restoration of tyrosine hydroxylase immunoreactive neurons and total cells in the nigrostriatal tract, improves the motor performance in MPTP-treated mice, and may serve as a therapeutic strategy for Parkinson's disease.

A dominant negative ERß splice variant determines the effectiveness of early or late estrogen therapy after ovariectomy in rats.

The molecular mechanisms for the discrepancy in outcome of initiating estrogen therapy (ET) around peri-menopause or several years after menopause in women are unknown. We hypothesize that the level of expression of a dominant negative estrogen receptor (ER) ß variant, ERß2, may be a key factor determining the effectiveness of ET in post-menopausal women. We tested this hypothesis in ovariectomized nine month-old (an age when irregular estrous cycles occur) female Sprague Dawley rats. Estradiol treatment was initiated either 6 days (Early ET, analogous to 4 months post-menopause in humans), or 180 days (Late ET, analogous to 11 years post-menopause in humans) after ovariectomy. Although ERß2 expression increased in all OVX rats, neurogenic and neuroprotective responses to estradiol differed in Early and Late ET. Early ET reduced ERß2 expression in both hippocampus and white blood cells, increased the hippocampal cell proliferation as assessed by Ki-67 expression, and improved mobility in the forced swim test. Late ET resulted in either no or modest effects on these parameters. There was a close correlation between the degree of ERß2 expression and the preservation of neural effects by ET after OVX in rats, supporting the hypothesis that persistent elevated levels of ERß2 are a molecular basis for the diminished effectiveness of ET in late post-menopausal women. The correlation between the expression of ERß2 in circulating white blood cells and brain cells suggests that ERß2 expression in peripheral blood cells may be an easily accessible marker to predict the effective window for ET in the brain.