Faculty Bibliography

Colony collapse disorder (CCD) is a multi-faceted syndrome decimating bee populations worldwide, and a group of viruses of the widely distributed Dicistroviridae family have been identified as a causing agent of CCD. This family of viruses employs non-coding RNA sequences, called internal ribosomal entry sites (IRESs), to precisely exploit the host machinery for viral protein production. Using single-particle cryo-electron microscopy (cryo-EM), we have characterized how the IRES of Israeli acute paralysis virus (IAPV) intergenic region captures and redirects translating ribosomes toward viral RNA messages. We reconstituted two in vitro reactions targeting a pre-translocation and a post-translocation state of the IAPV-IRES in the ribosome, allowing us to identify six structures using image processing classification methods. From these, we reconstructed the trajectory of IAPV-IRES from the early small subunit recruitment to the final post-translocated state in the ribosome. An early commitment of IRES/ribosome complexes for global pre-translocation mimicry explains the high efficiency observed for this IRES. Efforts directed toward fighting CCD by targeting the IAPV-IRES using RNA-interference technology are underway, and the structural framework presented here may assist in further refining these approaches.

Cellular senescence is a mechanism used by mitotic cells to prevent uncontrolled cell division. As senescent cells persist in tissues, they cause local inflammation and are harmful to surrounding cells, contributing to aging. Generally, neurodegenerative diseases, such as Parkinson's, are disorders of aging. The contribution of cellular senescence to neurodegeneration is still unclear. SATB1 is a DNA binding protein associated with Parkinson's disease. We report that SATB1 prevents cellular senescence in post-mitotic dopaminergic neurons. Loss of SATB1 causes activation of a cellular senescence transcriptional program in dopamine neurons both in human stem cell-derived dopaminergic neurons and in mice. We observed phenotypes that are central to cellular senescence in SATB1 knockout dopamine neurons in vitro and in vivo. Moreover, we found that SATB1 directly represses expression of the pro-senescence factor p21 in dopaminergic neurons. Our data implicate senescence of dopamine neurons as a contributing factor in the pathology of Parkinson's disease.

The apical surface of the terminally differentiated mammalian urothelial umbrella cell is mechanically stable and highly impermeable, in part due its coverage by urothelial plaques consisting of 2D-crystals of uroplakin particles. The mechanism for regulating the uroplakin/plaque level is unclear. We found that genetic ablation of the highly tissue-specific sorting nexin Snx31, which localizes to plaques lining the multivesicular bodies (MVBs) in urothelial umbrella cells, abolishes MVBs suggesting that Snx31 plays a role in stabilizing the MVB-associated plaques by allowing them to achieve a greater curvature. Strikingly, Snx31 ablation also induces a massive accumulation of uroplakin-containing mitochondria-derived lipid droplets (LDs), which mediate uroplakin degradation via autophagy/lipophagy, leading to the loss of apical and fusiform vesicle plaques. These results suggest that MVBs play an active role in suppressing the excessive/wasteful endocytic degradation of uroplakins. Failure of this suppression mechanism triggers the formation of mitochondrial LDs so that excessive uroplakin membranes can be sequestered and degraded. Since mitochondrial LD formation, which occurs at a low level in normal urothelium, can also be induced by disturbance in uroplakin polymerization due to individual uroplakin-knockout and by arsenite, a bladder carcinogen, this pathway may represent an inducible, versatile urothelial detoxification mechanism. [Media: see text] [Media: see text] [Media: see text].

Distinct oxygenases and their oxylipin products have been shown to participate in thermogenesis by mediating physiological adaptations required to sustain body temperature. Since the role of the lipoxygenase (LOX) family in cold adaptation remains elusive, we aimed to investigate whether, and how, LOX activity is required for cold adaptation and to identify LOX-derived lipid mediators that could serve as putative cold mimetics with therapeutic potential to combat diabetes. By utilizing mass-spectrometry-based lipidomics in mice and humans, we demonstrated that cold and β3-adrenergic stimulation could promote the biosynthesis and release of 12-LOX metabolites from brown adipose tissue (BAT). Moreover, 12-LOX ablation in mouse brown adipocytes impaired glucose uptake and metabolism, resulting in blunted adaptation to the cold in vivo. The cold-induced 12-LOX product 12-HEPE was found to be a batokine that improves glucose metabolism by promoting glucose uptake into adipocytes and skeletal muscle through activation of an insulin-like intracellular signaling pathway.

Background: Plakophilin-2 (PKP2) is classically defined as a protein of the desmosome, an intercellular adhesion structure that also acts as a signaling hub to maintain structural and electrical homeostasis. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events that occur consequent to its mutation. Here we sought to captureearly molecular/cellular events that can act as nascent substrates for subsequent arrhythmic/cardiomyopathic phenotypes.
Method(s): We used multiple quantitative imaging modalities, as well as biochemical and high-resolution mass spectrometry methods to study the functional/structural properties of cells/tissues derived from cardiomyocytespecific, tamoxifen-activated, PKP2 knockout mice ("PKP2cKO"). Studies were carried out 14 days post-tamoxifen injection, a time point preceding an overt electrical or structural phenotype.Myocytes from right or left ventricular free wall were studied separately, to detect functional/structural asymmetries.
Result(s): Most properties of PKP2cKO left ventricular (LV) myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+transients, increased frequency of spontaneous Ca2+release events, increased [Ca2+] in the cytoplasm and sarcoplasmic reticulum compartments, and dynamic Ca2+accumulation in mitochondria. In addition, RyR2 in RV presented enhanced sensitivity to Ca2+and preferential phosphorylation in a domain known to modulate Ca2+gating. RNAseq at 14 days post-TAM showed no relevant difference in transcript abundance between RV and LV, neither in control nor in PKP2cKO cells, suggesting that in the earliest stage, [Ca2+]i dysfunction is not transcriptional. Rather, we found an RV-predominant increase in membrane permeability that can permit Ca2+entry into the cell. Cx43 ablation mitigated the increase in membrane permeability, the accumulation of cytoplasmic Ca2+and the early stages of RV dysfunction.
Conclusion(s): Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy and that is, at least in part, mediated by a Cx43-dependent membrane conduit. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage

Determining the premalignant lesions that develop into malignant tumours remains a daunting task. Brain tumours are frequently characterised by a block in differentiation, implying that normal developmental pathways become hijacked during tumourigenesis. However, the heterogeneity of stem cells and their progenitors in the brain suggests there are many potential routes to tumour initiation. Studies in Drosophila melanogaster have enhanced our understanding of the tumourigenic potential of distinct cell types in the brain. Here we review recent studies that have improved our knowledge of neural stem cell behaviour during development and in brain tumour models.

Uterus transplantation is an emerging technology adding to the arsenal of treatments for infertility; specifically the only available treatment for uterine factor infertility. Ethical investigations concerning risks to uteri donors and transplant recipients have been discussed in the literature. However, missing from the discourse is the potential of uterus transplantation in other groups of genetically XY women who experience uterine factor infertility. There have been philosophical inquiries concerning uterus transplantation in genetically XY women, which includes transgender women and women with complete androgen insufficiency syndrome. We discuss the potential medical steps necessary and associated risks for uterus transplantation in genetically XY women. Presently, the medical technology does not exist to make uterus transplantation a safe and effective option for genetically XY women, however this group should not be summarily excluded from participation in trials. Laboratory research is needed to better understand and reduce medical risk and widen the field to all women who face uterine factor infertility.

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.

Impaired wound healing is a major complication of diabetes and despite the associated risks, treatment strategies for diabetic wounds remain limited. This is due, in part, to an incomplete understanding of the underlying pathological mechanisms, including the effects of hyperglycemia on components of the extracellular matrix (ECM). In the present study, we explored whether the expression of thrombospondin-2 (TSP2), a matricellular protein with a demonstrated role in response to injury, was associated with delayed healing in diabetes. First, we found that TSP2 expression was elevated in diabetic mice and skin from diabetic patients. Then, to determine the contribution of TSP2 to impaired healing in diabetes, we developed a novel diabetic TSP2-deficient model. Though the TSP2 deficient mice developed obesity and hyperglycemia comparable to diabetic control mice, they exhibited significantly improved healing, characterized by accelerated re-epithelialization and increased granulation tissue formation, fibroblast migration, and blood vessel maturation. We further found that hyperglycemia increased TSP2 expression in fibroblasts, the major cellular source of TSP2 in wounds. Mechanistically, high glucose increased activation of the hexosamine pathway and NF-κB signaling to elevate TSP2 expression. Our studies demonstrate that hyperglycemia-induced TSP2 expression contributes to impaired healing in diabetes.