Male seminal relaxin contributes to induction of the post-mating cytokine response in the female mouse uterus

The hormone relaxin is important in female reproduction for embryo implantation, cardiovascular function, and during labor and lactation. Relaxin is also synthesized in males by organs of the male tract. We hypothesized that relaxin might be one component of seminal plasma responsible for eliciting the female cytokine response induced in the uterus at mating. When recombinant relaxin was injected into the uterus of wild-type (Rln+/+) mice at estrus, it evoked the production of Cxcl1 mRNA and its secreted protein product CXCL1 in four of eight animals. Mating experiments were then conducted using mice with a null mutation in the relaxin gene (Rln−/− mice). qRT-PCR analysis of mRNA expression in wild-type females showed diminished uterine expression of several cytokine and chemokine genes in the absence of male relaxin. Similar differences were also noted comparing Rln−/− and Rln+/+ females mated to wild-type males. Quantification of uterine luminal fluid cytokine content confirmed that male relaxin provokes the production of CXCL10 and CSF3 in Rln+/+ females. Differences were also seen comparing Rln−/− and Rln+/+ females mated with Rln−/− males for CXCL1, CSF3, and CCL5, implying that endogenous relaxin in females might prime the uterus to respond appropriately to seminal fluid at coitus. Finally, pan-leukocyte CD45 mRNA was increased in wild-type matings compared to other combinations, implying that male and female relaxin may trigger leukocyte expansion in the uterus. We conclude that male and/or female relaxin may be important in activating the uterine cytokine/chemokine network required to initiate maternal immune adaptation to pregnancy

Speech Communication

Contains reports on five research projects.C.J. Lebel FellowshipNational Institutes of Health (Grant 5 T32 NS07040)National Institutes of Health (Grant 5 R01 NS04332)National Science Foundation (Grant 1ST 80-17599)U.S. Navy - Naval Electronic Systems Command Contract (N00039-85-C-0254)U.S. Navy - Naval Electronic Systems Command Contract (N00039-85-C-0341)U.S. Navy - Naval Electronic Systems Command Contract (N00039-85-C-0290

Testing the Limits of Antisense Oligonucleotide Treatment for Myotonic Dystrophy Type 1

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biomedical Genetics, 2016.Myotonic dystrophy type 1 (DM1) is a dominantly-inherited muscular dystrophy that leads to progressive disease of skeletal muscle, the cardiac conduction system (CCS), and the brain. DM1 is caused by a microsatellite CTG repeat expansion in the 3`-untranslated region of DMPK. The mutant DMPK (mutDMPK) is transcribed, producing expanded CUG-repeat mRNAs that are retained in nuclear foci. The CUG-repeat RNA has gain-of-function properties and is considered a primary driver of disease in DM1. In an effort to accelerate decay of toxic CUG-repeat RNA, DMPK-targeting antisense oligonucleotides (ASOs) were developed, and show robust activity in cardiac and skeletal muscle. Indeed, proof-of-concept experiments in mouse models of CUG-repeat RNA toxicity showed biochemical and phenotypic reversal with systemic ASO treatment. However, as with any knockdown strategy for a dominant disease, a key question remains whether collateral silencing of wild-type DMPK will be tolerated. This is particularly important for DM1, as nuclear-retained mutDMPK transcripts are not translated, and DMPK protein levels are reduced by 50% in DM1 tissues. Importantly, it was previously suggested that the CCS is sensitive to DMPK dose, as both heterozygous and homozygous Dmpk knockout mice were reported to show CCS slowing. Furthermore, homozygous Dmpk knockout mice were reported to develop skeletal myopathy. This raises the possibility that CUG-repeat RNA may not be the only driver of disease in DM1, and the risk of DMPK reduction may be a contraindication for antisense knockdown therapy. To circumvent this concern, we first considered a method to selectively target mutDMPK mRNA over wild-type. We hypothesized that mutDMPK transcripts may be hyper-sensitive to ASO-targeting, due to their increased nuclear co-residence with the ASO-directed RNA endonuclease, RNase-H1. Using a transgenic mouse model that produces two alleles, one with an expanded CTG repeat (HSAXLR) and the other without (HSANR), we tested this hypothesis. Importantly, both alleles accumulate similar levels of mRNA in skeletal muscle, but only HSAXLR mice develop CUG-repeat RNA nuclear foci and DM1 biochemical and physiologic phenotypes. Nevertheless, subcutaneous injection of an ASO that targets sequence found in both alleles led to similar reductions of HSAXLR and HSANR mRNA in skeletal muscle. This suggests that selective-targeting of mutDMPK may not be feasible using the current highly-potent ASOs. Therefore, we focused on re-examining the pathophysiological role of DMPK loss in the CCS and skeletal muscle. Using Dmpk knockout mice we investigated the consequences of Dmpk deletion on cardiac and skeletal muscle physiologic function. Furthermore, to model the effects of ASO silencing in DM1 patients, we examined cardiac and skeletal muscle function in heterozygous Dmpk knockouts with long-term ASO treatment. Measurements of cardiac conduction intervals, cardiac contractile function, and skeletal muscle function were performed in mice on two genetic backgrounds, up to 18 months in age. Contrary to previous reports, we found that both complete absence of Dmpk protein or long-term reduction (>90%) by ASOs was well-tolerated, and did not significantly impact cardiac or skeletal muscle structure or function. These findings are encouraging for the development of ASO therapeutics for DM1, and suggest that collateral DMPK reduction will be tolerated. Lastly, the current antisense drugs for DM1 direct an RNase-H1 cleavage of DMPK upstream of the CUG repeat tract. Successful CUG-repeat RNA reduction, therefore, relies on the cellular RNA decay machinery to process through the expanded CUG repeat tract. It is unclear how efficient this clearance will be, and current methods to measure CUG-repeat RNA are limited due to background signal and incompatibility with variable repeat lengths. Therefore, we developed an assay to quantify CUG-repeat RNA mass, called Repeat Mass Assay (RMA), by reverse transcribing directly from the repeat tract. RMA shows excellent correlation (R2 = 0.99) with repeat RNA mass predictions in animal models. Furthermore, following ASO treatment, RMA measurements show excellent correlation (R2 = 0.99) with transgene knockdown measured by quantitative reverse transcriptase PCR. This validates the accuracy and utility of RMA, and suggests that in DM1 mouse models CUG-repeat decay is complete. In conclusion, while selective targeting of mutDMPK may not be feasible, ASO targeting is likely to lead to effective clearance of toxic CUG-repeat RNA. Additionally, the mouse CCS is not sensitive to Dmpk dose, and collateral reduction of DMPK protein is unlikely to exacerbate disease phenotypes. These findings are encouraging and give basic insight into DM1 mechanisms, and are critical for translation of gene-silencing therapy for DM1

A fiber optic array spectrometer with parallel multichannel optical lock-in detection and its application to in situ lighting measurements

Lock-in detection is a well-established technique to measure a modulated electrical signal in the presence of noise and large background signals. Commercially available lock-in amplifiers are not adapted to detector arrays such as used in modern compact optical spectrometers. In this case, performing lock-in detection requires a parallel multichannel processing of all spectral channels simultaneously. This paper describes an optical lock-in spectrometer featuring parallel and multichannel lock-in detection. It is based on two compact array spectrometers working in phase quadrature thanks to a wideband optical modulator assembled using standard optical components. In the application example, irradiance and illuminance lock-in measurements were performed on commercial LED lamps exhibiting temporal light modulation at different frequencies. A remote sensing optical device was designed to measure from a distance the temporal luminous waveform of a specific LED lamp and send it to the reference input of the optical lock-in spectrometer. When these lamps were operating together on top of a large continuous luminous background up to 30 times more intense, the optical lock-in spectrometer successfully retrieved the respective spectral irradiance distributions and color parameters of each lamp, allowing their characterization to be performed independently of the other light sources illuminating the sensor during the measurements.</p

Nox4 NADPH oxidase contributes to smooth muscle cell phenotypes associated with unstable atherosclerotic plaques

Plaque instability associated with acute coronary syndromes results in part from apoptosis and senescence of cells within the atherosclerotic (AS) lesion. Increased cellular oxidative stress has been proposed to contribute to plaque progression and changes in composition, leading to plaque instability. Our objective was to examine the role of NADPH oxidase in smooth muscle cell (SMC) phenotypes associated with an unstable plaque. Aortae were isolated from pre-lesion (8 weeks of age) and post-lesion (35 weeks of age) hypercholesterolemic mice (ApoE−/−/LDLR−/−, AS), and age-matched normal C57BL/6J mice. We observed an age-dependent increase in reactive oxygen species (ROS) in aorta from AS mice, with evidence for elevated ROS prior to lesion development. Whereas macrophage infiltration was restricted to the lesion, oxidized lipids extended beyond the plaque and into the vessel wall. Consistent with these findings, we observed dynamic changes in the expression of NADPH oxidases in AS vessels. Specifically, Nox1 expression was increased early and decreased with lesion progression, while induction of Nox4 was a late event. Nox2 and p22phox were elevated throughout lesion development. Similar to observations in aortae, SMCs isolated from the lesion of AS aortae had decreased Nox1 and increased Nox4 levels as compared to SMCs from normal mice. AS SMCs demonstrated increased generation of ROS, cell cycle arrest, evidence of senescence, and increased susceptibility to apoptosis. Overexpression of Nox4 in normal SMCs recapitulated the phenotypes of the AS SMCs. We conclude that increased expression of Nox4 in AS may drive SMC phenotypes that lead to the plaque instability and rupture responsible for myocardial infarction and stroke

Reducing Levels of Toxic RNA with Small Molecules

Myotonic dystrophy (DM) is one of the most common forms of muscular dystrophy. DM is an autosomal dominant disease caused by a toxic gain of function RNA. The toxic RNA is produced from expanded noncoding CTG/CCTG repeats, and these CUG/CCUG repeats sequester the Muscleblind-like (MBNL) family of RNA binding proteins. The MBNL proteins are regulators of alternative splicing, and their sequestration has been linked with mis-splicing events in DM. A previously reported screen for small molecules found that pentamidine was able to improve splicing defects associated with DM. Biochemical experiments and cell and mouse model studies of the disease indicate that pentamidine and related compounds may work through binding the CTG*CAG repeat DNA to inhibit transcription. Analysis of a series of methylene linker analogues of pentamidine revealed that heptamidine reverses splicing defects and rescues myotonia in a DM1 mouse model