Faculty Bibliography

Germline pathogenic variants in BRCA1 confer a high risk of developing breast and ovarian cancer. The BRCA1 exon 11 (formally exon 10) is one of the largest exons and codes for the nuclear localization signals of the corresponding gene product. This exon can be partially or entirely skipped during pre-mRNA splicing, leading to three major in-frame isoforms that are detectable in most cell types and tissue, and in normal and cancer settings. However, it is unclear whether the splicing imbalance of this exon is associated with cancer risk. Here we identify a common genetic variant in intron 10, rs5820483 (NC_000017.11:g.43095106_43095108dup), which is associated with exon 11 isoform expression and alternative splicing, and with the risk of breast cancer, but not ovarian cancer, in BRCA1 pathogenic variant carriers. The identification of this genetic effect was confirmed by analogous observations in mouse cells and tissue in which a loxP sequence was inserted in the syntenic intronic region. The prediction that the rs5820483 minor allele variant would create a binding site for the splicing silencer hnRNP A1 was confirmed by pull-down assays. Our data suggest that perturbation of BRCA1 exon 11 splicing modifies the breast cancer risk conferred by pathogenic variants of this gene.

PURPOSE/OBJECTIVE:To evaluate whether young women with idiopathic early ovarian aging, as defined by producing fewer oocytes than expected for a given age over multiple in vitro fertilization (IVF) cycles, have changes in telomere length and epigenetic age indicating accelerated biological aging (i.e., increased risk of morbidity and mortality). METHODS:A prospective cohort study was conducted at two Danish public fertility clinics. A total of 55 young women (≤ 37 years) with at least two IVF cycles with ≤ 5 harvested oocytes despite sufficient stimulation with follicle-stimulating hormone (FSH) were included in the early ovarian aging group. As controls, 52 young women (≤ 37 years) with normal ovarian function, defined by at least eight harvested oocytes, were included. Relative telomere length (rTL) and epigenetic age acceleration (AgeAccel) were measured in white blood cells as markers of premenopausal accelerated biological aging. RESULTS:rTL was comparable with a mean of 0.46 (± SD 0.12) in the early ovarian aging group and 0.47 (0.14) in the normal ovarian aging group. The AgeAccel of the early ovarian aging group was, insignificantly, 0.5 years older, but this difference disappeared when adjusting for chronological age. Sub-analysis using Anti-Müllerian hormone (AMH) as selection criterion for the two groups did not change the results. CONCLUSION/CONCLUSIONS:We did not find any indications of accelerated aging in whole blood from young women with idiopathic early ovarian aging. Further investigations in a similar cohort of premenopausal women or other tissues are needed to fully elucidate the potential relationship between premenopausal accelerated biological aging and early ovarian aging.

Barth syndrome is a rare X-linked multisystem mitochondrial disease that is caused by variants in the tafazzin gene leading to deficient and abnormal cardiolipin. Previous research has focused on the cardiomyopathy and neutropenia in individuals with Barth syndrome, yet just as common are the least explored neurological aspects of Barth syndrome. This review focuses on the major neuropsychological and neurophysiological phenotypes that affect the quality of life of individuals with Barth syndrome, including difficulties in sensory perception and feeding, fatigue, and cognitive and psychological challenges. We propose selected pathogenetic mechanisms underlying these phenotypes and draw parallels to other relevant disorders. Finally, avenues for future research are also suggested.

Rhodopsins are light-detecting proteins coupled with retinal chromophores essential for visual function. Coincidentally, dysfunctional rhodopsin homeostasis underlies retinal degeneration in humans and model organisms. Drosophila ninaEG69D mutant is one such example, where the encoded Rh1 protein imposes endoplasmic reticulum (ER) stress and causes light-dependent retinal degeneration. The underlying reason for such light-dependency remains unknown. Here, we report that Drosophila fatty acid binding protein (fabp) is a gene induced in ninaEG69D/+ photoreceptors, and regulates light-dependent Rhodopsin-1 (Rh1) protein clearance and photoreceptor survival. Specifically, our photoreceptor-specific gene expression profiling study in ninaEG69D/+ flies revealed increased expression of fabp together with other genes that control light-dependent Rh1 protein degradation. fabp induction in ninaEG69D photoreceptors required vitamin A and its transporter genes. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. The increase in Rh1 levels under these conditions was dependent on Arrestin2 that mediates feedback inhibition of light-activated Rh1. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-induced Rh1 degradation. These observations reveal a previously unrecognized link between light-dependent Rh1 proteostasis and the ER-stress imposing ninaEG69D mutant that cause retinal degeneration.

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, four distinct nucleotide-binding sites, and conformational dynamics pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S. cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with functional assays of dynein motility and mutagenesis studies, suggest that the compounds inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein's main catalytic site in the AAA1 domain. A double Walker B mutation of the AAA3 and AAA4 sites substantially reduces enzyme activity, suggesting that targeting these regulatory domains is sufficient to inhibit dynein. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein's AAA domains.

Mitochondrial cristae are extraordinarily crowded with proteins, which puts stress on the bilayer organization of lipids. We tested the hypothesis that the high concentration of proteins drives the tafazzin-catalyzed remodeling of fatty acids in cardiolipin, thereby reducing bilayer stress in the membrane. Specifically, we tested whether protein crowding induces cardiolipin remodeling and whether the lack of cardiolipin remodeling prevents the membrane from accumulating proteins. In vitro, the incorporation of large amounts of proteins into liposomes altered the outcome of the remodeling reaction. In yeast, the concentration of proteins involved in oxidative phosphorylation (OXPHOS) correlated with the cardiolipin composition. Genetic ablation of either remodeling or biosynthesis of cardiolipin caused a substantial drop in the surface density of OXPHOS proteins in the inner membrane of the mouse heart and Drosophila flight muscle mitochondria. Our data suggest that OXPHOS protein crowding induces cardiolipin remodelling and that remodeled cardiolipin supports the high concentration of these proteins in the inner mitochondrial membrane.

Inflammatory Bowel Disease (IBD) is an autoimmune condition with complicated pathology and diverse clinical signs. TNFα is believed to play a crucial role in the pathogenesis of IBD. We recently identified fexofenadine, a well-known antagonist of histamine H1 receptor, as a novel inhibitor of TNFα signaling. Additionally, cytosolic phospholipase A2 (cPLA2) was isolated as a binding target of fexofenadine, and fexofenadine-mediated anti-TNF activity relied on cPLA2 in vitro. The objective of this study is to determine whether fexofenadine is therapeutic against chemically-induced murine IBD model and whether cPLA2 and/or histamine H1 receptor is important for fexofenadine's anti-inflammatory activity in vivo by leveraging various genetically modified mice and chemically induced murine IBD models. Both dextran sulfate sodium- and 2, 4, 6-trinitrobenzene sulfonic acid-induced murine IBD models revealed that orally delivered fexofenadine was therapeutic against IBD, evidenced by mitigated clinical symptoms, decreased secretions of the proinflammatory cytokine IL-6 and IL-1β, lowered intestinal inflammation, and reduced p-p65 and p-IĸBα. Intriguingly, Fexofenadine-mediated protective effects against IBD were lost in cPLA2 deficient mice but not in histamine H1 receptor-deficient mice. Collectively, these findings demonstrate the therapeutic effects of over-the-counter drug Fexofenadine in treating DSS-induced IBD murine and provide first in vivo evidence showing that cPLA2 is required for fexofenadine's therapeutic effects in murine IBD model and probably other inflammatory and autoimmune diseases as well.

Gpr125, encoded by Adgra3, is an orphan adhesion G-protein coupled receptor (aGPCR) implicated in modulating Wnt signaling and planar polarity. Here we establish both physiological and pathological roles for Gpr125. We show that mice lacking Gpr125 or its signaling domains display an ocular phenotype with many hallmarks of human dry eye syndrome. These include squinting, abnormal lacrimation, mucus accumulation, swollen eyelids and inflammatory infiltration of lacrimal and meibomian glands. Utilizing a Gpr125-β-gal reporter and scRNAseq, we identify Gpr125 expression in a discrete population of cells located at the tips of migrating embryonic lacrimal ducts. By lineage tracing we show these cells function as progenitors of the adult lacrimal myoepithelium. Beyond defining an essential role for Gpr125 in tear film and identifying its utility as a marker of lacrimal progenitors, this study implicates Gpr125 in the etiology of blepharitis and dry eye syndrome, and defines novel animal models of these common maladies

PIWI proteins and their guiding Piwi-interacting small RNAs (piRNAs) are crucial for fertility and transposon defense in the animal germline. In most species, the majority of piRNAs are produced from distinct large genomic loci, called piRNA clusters. It is assumed that germline-expressed piRNA clusters, particularly in Drosophila, act as principal regulators to control transposons dispersed across the genome. Here, using synteny analysis, we show that large clusters are evolutionarily labile, arise at loci characterized by recurrent chromosomal rearrangements, and are mostly species-specific across the Drosophila genus. By engineering chromosomal deletions in D. melanogaster, we demonstrate that the three largest germline clusters, which account for the accumulation of >40% of all transposon-targeting piRNAs in ovaries, are neither required for fertility nor for transposon regulation in trans. We provide further evidence that dispersed elements, rather than the regulatory action of large Drosophila germline clusters in trans, may be central for transposon defense.

PERK is an endoplasmic reticulum (ER) transmembrane sensor that phosphorylates eIF2α to initiate the Unfolded Protein Response (UPR). eIF2α phosphorylation promotes stress-responsive gene expression most notably through the transcription factor ATF4 that contains a regulatory 5' leader. Possible PERK effectors other than ATF4 remain poorly understood. Here, we report that the bZIP transcription factor Xrp1 is required for ATF4-independent PERK signaling. Cell-type-specific gene expression profiling in Drosophila indicated that delta-family glutathione-S-transferases (gstD) are prominently induced by the UPR-activating transgene Rh1^G69D. Perk was necessary and sufficient for such gstD induction, but ATF4 was not required. Instead, Perk and other regulators of eIF2α phosphorylation regulated Xrp1 protein levels to induce gstDs. The Xrp1 5' leader has a conserved upstream Open Reading Frame (uORF) analogous to those that regulate ATF4 translation. The gstD-GFP reporter induction required putative Xrp1 binding sites. These results indicate that antioxidant genes are highly induced by a previously unrecognized UPR signaling axis consisting of PERK and Xrp1.