Faculty Bibliography

Primordial germ cells (PGCs) in many species associate intimately with endodermal cells, but the significance of such interactions is largely unexplored. Here, we show that Caenorhabditis elegans PGCs form lobes that are removed and digested by endodermal cells, dramatically altering PGC size and mitochondrial content. We demonstrate that endodermal cells do not scavenge lobes PGCs shed, but rather, actively remove lobes from the cell body. CED-10 (Rac)-induced actin, DYN-1 (dynamin) and LST-4 (SNX9) transiently surround lobe necks and are required within endodermal cells for lobe scission, suggesting that scission occurs through a mechanism resembling vesicle endocytosis. These findings reveal an unexpected role for endoderm in altering the contents of embryonic PGCs, and define a form of developmentally programmed cell remodelling involving intercellular cannibalism. Active roles for engulfing cells have been proposed in several neuronal remodelling events, suggesting that intercellular cannibalism may be a more widespread method used to shape cells than previously thought.

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.

Glucocorticoid resistance is a risk factor for Alzheimer's disease (AD). Molecular and cellular mechanisms of glucocorticoid resistance in the brain have remained unknown and are potential therapeutic targets. Phosphorylation of glucocorticoid receptors (GR) by brain-derived neurotrophic factor (BDNF) signaling integrates both pathways for remodeling synaptic structure and plasticity. The goal of this study is to test the role of the BDNF-dependent pathway on glucocorticoid signaling in a mouse model of glucocorticoid resistance. We report that deletion of GR phosphorylation at BDNF-responding sites and downstream signaling via the MAPK-phosphatase DUSP1 triggers tau phosphorylation and dendritic spine atrophy in mouse cortex. In human cortex, DUSP1 protein expression correlates with tau phosphorylation, synaptic defects and cognitive decline in subjects diagnosed with AD. These findings provide evidence for a causal role of BDNF-dependent GR signaling in tau neuropathology and indicate that DUSP1 is a potential target for therapeutic interventions.

Marfan syndrome (MFS) is a multi-system connective tissue disorder that results from mutations to the gene that codes the elastin-associated glycoprotein fibrillin-1. Although elastic fibers are compromised throughout the arterial tree, the most severe phenotype manifests in the ascending aorta. By comparing biaxial mechanics of the ascending and descending thoracic aorta in a mouse model of MFS, we show that aneurysmal propensity correlates well with both a marked increase in circumferential material stiffness and an increase in intramural shear stress despite a near maintenance of circumferential stress. This finding is corroborated via a comparison of the present results with previously reported findings for both the carotid artery from the same mouse model of MFS and for the thoracic aorta from another model of elastin-associated glycoprotein deficiency that does not predispose to thoracic aortic aneurysms. We submit that the unique biaxial loading of the ascending thoracic aorta conspires with fibrillin-1 deficiency to render this aortic segment vulnerable to aneurysm and rupture.

OBJECTIVES: Obesity and obesity-associated inflammation is central to a variety of end-organ sequelae including atherosclerosis, a leading cause of death worldwide. Although mouse models have provided important insights into the immunopathogenesis of various diseases, modeling atherosclerosis in mice has proven difficult. Specifically, wild-type (WT) mice are resistant to developing atherosclerosis, while commonly used genetically modified mouse models of atherosclerosis are poor mimics of human disease. The lack of a physiologically relevant experimental model of atherosclerosis has hindered the understanding of mechanisms regulating disease development and progression as well as the development of translational therapies. Recent evidence suggests that housing mice within their thermoneutral zone profoundly alters murine physiology, including both metabolic and immune processes. We hypothesized that thermoneutral housing would allow for augmentation of atherosclerosis induction and progression in mice. METHODS: ApoE-/- and WT mice were housed at either standard (TS) or thermoneutral (TN) temperatures and fed either a chow or obesogenic "Western" diet. Analysis included quantification of (i) obesity and obesity-associated downstream sequelae, (ii) the development and progression of atherosclerosis, and (iii) inflammatory gene expression pathways related to atherosclerosis. RESULTS: Housing mice at TN, in combination with an obesogenic "Western" diet, profoundly augmented obesity development, exacerbated atherosclerosis in ApoE-/- mice, and initiated atherosclerosis development in WT mice. This increased disease burden was associated with altered lipid profiles, including cholesterol levels and fractions, and increased aortic plaque size. In addition to the mild induction of atherosclerosis, we similarly observed increased levels of aortic and white adipose tissue inflammation and increased circulating immune cell expression of pathways related to adverse cardiovascular outcome. CONCLUSIONS: In sum, our novel data in WT C57Bl/6 mice suggest that modulation of a single environmental variable, temperature, dramatically alters mouse physiology, metabolism, and inflammation, allowing for an improved mouse model of atherosclerosis. Thus, thermoneutral housing of mice shows promise in yielding a better understanding of the cellular and molecular pathways underlying the pathogenesis of diverse diseases.

Implantation rate decreases and miscarriage rate increases with advancing maternal age. The oocyte must be the locus of reproductive aging because donation of oocytes from younger to older women abrogates the effects of aging on fecundity. Nuclear transfer experiments in a mouse model of reproductive aging show that the reproductive aging phenotype segregates with the nucleus rather than the cytoplasm. A number of factors within the nucleus have been hypothesized to mediate reproductive aging, including disruption of cohesions, reduced chiasma, aneuploidy, disrupted meiotic spindles, and DNA damage caused by chronic exposure to reactive oxygen species. We have proposed telomere attrition as a parsimonious way to explain these diverse effects of aging on oocyte function. Telomeres are repetitive sequences of DNA and associated proteins, which form a loop (t loop) at chromosome ends. Telomeres prevent the blunt end of DNA from triggering a DNA damage response. Previously, we showed that experimental telomere shortening phenocopies reproductive aging in mice. Telomere shortening causes reduced synapsis and chiasma, chromosome fusions, embryo arrest and fragmentation, and abnormal meiotic spindles. Telomere length of polar bodies predicts the fragmentation of human embryos. Telomerase, the reverse transcriptase capable of reconstituting shortened telomeres, is only minimally active in oocytes and preimplantation embryos. Intriguingly, during the first cell cycles following activation, telomeres robustly elongate via a DNA double-strand break mechanism called alternative lengthening of telomeres (ALTs). Alternative lengthening of telomere takes place even in telomerase-null mice. This mechanism of telomere elongation previously had been found only in cancer cells lacking telomerase activity. We propose that ALT elongates telomeres across generations but does so at the cost of extensive genomic instability in preimplantation embryos.

OBJECTIVE: Telomere attrition may mediate some of the effects of aging on reproductive function in women. Mice null or haploinsufficient for telomerase phenocopy the profile of reproductive aging in women, with progressive infertility caused by numerous defects in their reproductive cells. Telomeropathies, such as Dyskeratosis Congenita, provide a natural experiment to test the Telomere Theory of Reproductive Aging in women. This study attempts to extensively characterize the reproductive function in women with telomeropathies for the first time. DESIGN: Blood samples, cumulus cells, and arrested embryos were collected following the cycle of A 30 year old woman with a precocious aging syndrome and aplastic anemia, attributed to a telomeropathy (AMH=0.3 and AFC=8). She underwent, controlled ovarian stimulation with E2 prime protocol and 600 IU/day of gonadotropin, using mixed protocol and GnRH antagonist for 18 days. MATERIALS AND METHODS: Monochrome multiplex quantitative polymerase chain reaction (qPCR) assay (Cawthon 2009) measured telomere length in leukocytes extracted from whole blood as well as, cumulus cells stripped from retrieved follicles. Telomere (T) amplification was normalized to a single copy gene (S), resulting in a T/S ratio proportional to average telomere length in the population. Single-Cell Amplification of Telomere Repeats (SCATR) PCR (Wang 2013) was used to measure telomere length in discarded embryo blastomeres. Telomere (T) amplification was normalized a reference gene (R), producing a T/R ratio . One-Way ANOVA test was used to determine statistical significance. RESULTS: Hyperstimulation resulted in only 7 oocytes and 1 euploid blastocyst. Over the treatment course, leukocyte telomere length increased from T/S ratio= 0.192+/-.0157 to 0.234+/-.0306 and there was a statistically significant (p= .0256) linear increase during the treatment. Further, telomere length in a retrieved parthenogenetic, 2-cell embryo was (T/R average= .169+/-.021) and that in cumulus cells (T/S= 0.586+/-.0147). Telomere lengths in all assayed cell types were shorter than those from age matched controls. CONCLUSIONS: Young woman with reduced ovarian reserve, poor response to ovarian stimulation and a high percentage of arrested embryos and aneuploid embryos was still able to generate one euploid blastocyst with high dose controlled ovarian stimulation, demonstrating the promise of ART for fertility preservation in women with telomeropathies. Intriguingly, controlled ovarian hyperstimulation increased her leukocyte telomere length. Presumably, the supraphysiologic levels of estrogen activated telomerase activity, consistent with prior studies reporting an estrogen response element in the TERT gene. Future studies should examine whether women with telomeropathies may benefit from estrogen supplementation

OBJECTIVE: The architecture and structure of the meiotic spindle influence embryo development(1) and risk of aneuploidy in women(2). The factors that disrupt the meiotic spindle remain incompletely understood. Dysfunctional mitochondria produce reactive oxygen species (ROS), which may directly perturb spindles(3). ROS also can disrupt spindles by inducing telomere attrition, since telomeres are essential for spindle formation and are especially susceptible to ROS(4). We studied the impact of ROS, produced by uncoupling mitochondria, on the area and retardance (measure ofmolecular order) ofmeiotic spindles and on mean telomere length of individual human oocytes. DESIGN: Prospective, randomized, paired research laboratory intervention. MATERIALS AND METHODS: 44 germinal vesicle (GV) stage oocytes were accessioned from 16 patients undergoing IVF/ICSI. GVs from each patient were randomly assigned to control or treatment group. Oocytes in the treatment group were cultured in media containing Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 750nM) for one hour to induce mitochondrial stress. Control oocytes were cultured in media without FCCP. GVoocytes from both groups were further cultured to permit meiotic maturation, as indicated by extrusion of the first polar body. Spindles were imaged non-invasively with an orientation independent polarized light microscope (Oosight, Hamilton Thorne, MA, USA). Spindle area and mean retardance were measured with software from the Oosight Imaging System. Mean oocyte telomere length was measured by single cell qPCR. Data were analyzed by Chi-square test and independent t test. RESULTS: Maturation rates of oocytes in the control and FCCP groups were 66.67% and 56.52% respectively. 71.4% of MII oocytes in the control group and 61.5% in the FCCP group had a detectable birefrigent spindle. FCCP decreased the area of the spindles compared to controls (19.84+/-2.11 sq. microns versus 37.77+/-4.79, P=0.011). Mean spindle retardance in the FCCP group also was significantly lower than that of controls (1.45+/-0.11 nm versus 2.19+/-0.16 nm, P=0.003). Telomere length (T/R ratio) did not differ between treatment and control groups (1.11+/-1.56 versus 1.29+/-0.19, P>0.05). Telomere length of oocytes with and without birefringence spindle within the treatment group also did not differ significantly (P>0.05). CONCLUSIONS: Meiotic maturation itself is relatively resistant to ROS, consistent with prior studies showing limited cell cycle check point control during oogenesis. However, ROS produced by acute mitochondrial stress disrupts spindle retardance and size. Reactive oxygen, at least acutely, does not induce telomere attrition in human oocytes