Faculty Bibliography

A dysfunctional endosomal pathway and abnormally enlarged early endosomes in neurons are an early characteristic of Down syndrome (DS) and Alzheimer's disease (AD). We have hypothesized that endosomal material can be released by endosomal multivesicular bodies (MVBs) into the extracellular space via exosomes to relieve neurons of accumulated endosomal contents when endosomal pathway function is compromised. Supporting this, we found that exosome secretion is enhanced in the brains of DS patients and a mouse model of the disease, and by DS fibroblasts. Furthermore, increased levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Importantly, CD63 knockdown diminished exosome release and worsened endosomal pathology in DS fibroblasts. Taken together, these data suggest that increased CD63 expression enhances exosome release as an endogenous mechanism mitigating endosomal abnormalities in DS. Thus, the upregulation of exosome release represents a potential therapeutic goal for neurodegenerative disorders with endosomal pathology.

The recent discovery of metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis has expanded the functions of this classically known mitogen. To dissect the molecular basis for this functional pleiotropy, we engineered an FGF1 partial agonist carrying triple mutations (FGF1DeltaHBS) that diminished its ability to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1DeltaHBS exhibited a severely reduced proliferative potential, while preserving the full metabolic activity of wild-type FGF1 in vitro and in vivo. Hence, suboptimal FGFR activation by a weak FGF1-FGFR dimer is sufficient to evoke a metabolic response, whereas full FGFR activation by stable and sustained dimerization is required to elicit a mitogenic response. In addition to providing a physical basis for the diverse activities of FGF1, our findings will impact ongoing drug discoveries targeting FGF1 and related FGFs for the treatment of a variety of human diseases.

Objective: We sought to assess our experience between a private (TH) and a public hospital (BH), both staffed by faculty and trainees of the same major university medical center. Methods: Our gastric cancer database was used to identify patients undergoing curative intent resection. Descriptive statistics were used to compare demographic data. Kaplan-Meier survival analysis was used to examine recurrence (RFS) and overall survival (OS). Multivariate proportional hazards regression was used to identify factors associated with RFS and OS. Data were risk - and disease stage-stratified. Results: There were 100 patients in the BH group and 242 in the TH group, with a median age 55 and 70.5 years respectively (p<0.001). The majority of BH patients were Asians (60, 60%), and Caucasians (172, 72.3%) in the TH group. The median number of days from diagnosis to surgical intervention at BH was 47.5 vs. 30 days in the TH (p=0.01). BH group had a smaller BMI and more frequently received distal or subtotal gastrectomy. Perioperative morbidity and mortality was equally distributed, as was 30-day readmission rate. Pathologic staging was similarly distributed. By multivariate analysis, hospital of treatment was not associated with RFS (p=0.48) nor OS (p=0.56). Conclusions: Patients receiving care for gastric cancer at major public hospitals have equally good clinical outcomes when compared to patients treated at private hospitals, if cared for by physicians within the same institution dedicated to disease specific entities. Overall survival by treatment hospital

The function of protein products generated from intramembraneous cleavage by the gamma-secretase complex is not well defined. The gamma-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein which results in Abeta, a transmembrane (TM) peptide. Another protein that undergoes a very similar gamma-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of the tyrosine kinase receptor B (TrkB). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time- dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Since this residue is immediately after the gamma-secretase cleavage site, we then examined if the p75(alphagamma) peptide, which is a product of both alpha- and gamma- cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether our results suggest that TrkB activation by p75(alphagamma) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimers disease, and epilepsy.

The developmental accumulation of proliferative germ cells in the C. elegans hermaphrodite is sensitive to the organismal environment. Previously, we found that the TGFbeta signaling pathway links the environment and proliferative germ cell accumulation. Neuronal DAF-7/TGFbeta causes a DAF-1/TGFbetaR signaling cascade in the gonadal distal tip cell (DTC), the germline stem cell niche, where it negatively regulates a DAF-3 SMAD and DAF-5 Sno-Ski. LAG-2, a founding DSL ligand family member, is produced in the DTC and activates the GLP-1/Notch receptor on adjacent germ cells to maintain germline stem cell fate. Here, we show that DAF-7/TGFbeta signaling promotes expression of lag-2 in the DTC in a daf-3-dependent manner. Using ChIP and one-hybrid assays, we find evidence for direct interaction between DAF-3 and the lag-2 promoter. We further identify a 25 bp DAF-3 binding element required for the DTC lag-2 reporter response to the environment and to DAF-7/TGFbeta signaling. Our results implicate DAF-3 repressor complex activity as a key molecular mechanism whereby the environment influences DSL ligand expression in the niche to modulate developmental expansion of the germline stem cell pool.

The Integrated Stress Response (ISR) refers to a signaling pathway initiated by stress-activated eIF2alpha kinases. Once activated, the pathway causes attenuation of global mRNA translation while also paradoxically inducing stress response gene expression. A detailed analysis of this pathway has helped us better understand how stressed cells coordinate gene expression at translational and transcriptional levels. The translational attenuation associated with this pathway has been largely attributed to the phosphorylation of the translational initiation factor eIF2alpha. However, independent studies are now pointing to a second translational regulation step involving a downstream ISR target, 4E-BP, in the inhibition of eIF4E and specifically cap-dependent translation. The activation of 4E-BP is consistent with previous reports implicating the roles of 4E-BP resistant, Internal Ribosome Entry Site (IRES) dependent translation in ISR active cells. In this review, we provide an overview of the translation inhibition mechanisms engaged by the ISR and how they impact the translation of stress response genes.

Study question: What happens to telomere length during human meiotic maturation and pre-implantation development? What is the impact of DNA damage and telomere attrition on human development? Summary answer: DNA damage and telomere attrition limit oocyte maturation in vitro. Telomeres are short in oocytes, increase markedly, and develop increased heterogeneity during pre-implantation development. What is known already: Telomere length reflects aging in many cell types. Sperm, which emerge from spermatogonia throughout the life of the man, have long telomeres. Oocytes from mouse and women maintain short telomeres. Telomerase, the enzyme that lengthens telomeres, is minimally active in mouse and human oocytes and pre-implantation embryos until blastocyst stage. Lacking appreciable telomerase activity, early mouse embryos elongate telomeres via Alternative Lengthening of Telomeres (ALT), which provides robust telomere elongation, but increased genomic instability. We do not know whether ALT occurs during early human development, and if so, when it takes place. Study design, size, duration: 60 immature germinal vesicle (GV) and metaphase I (M1) human oocytes donated to research from women ages 18-45 were collected and in vitro matured (IVM) for up to 48 hours following the subjects' retrieval. Additionally, 28 cryopreserved human embryos donated to research from 7 couples (age 27-42 years old), at the New York University Langone Fertility Center. Participants/materials, setting, methods: Immature oocytes were in vitro matured to metaphase II (M2). Frozen embryos between the 2 pronuclear (2PN) and day 3 (8-10-cell) stage were thawed and dissociated into single blastomere, intact blastocysts were processed whole. Telomere length was evaluated using Single Cell Amplification of Telomere Repeats PCR (SCATRPCR), expressed as a telomere to reference gene ratio (T/R ratio). DNA damage was assessed by immunoflorescent staining. Statistical analysis was performed using one-way ANOVA or T-test where appropriate. Main results and the role of chance: During oocyte maturation, immunostaining revealed that oocytes which arrested at the GV stage and failed to mature, contained robust and abundant DNA damage signaling on their chromosomes compared with successfully matured M2s (mean total florescent units = 35073.4 +/- 10051.8 vs. 843.7 +/- 74.9). Telomere length however did not differ significantly between arrested GV and M2 oocytes (mean T/R ratio = 0.074 +/- 0.040 vs 0.105 +/- 0.067). Telomere length increased significantly (p < 0.05) between M2 oocytes and both 2PN embryos (mean T/R ratio = 0.837 +/- 0.546) and blastocysts (mean T/R ratio = 0.634 +/- 0.260). The most significant elongation (p < 0.0002) occurred by day 2 (2-4 cell) (mean T/ R ratio = 0.957 +/- 767). Between day 2 and day 3 (mean T/R ratio = 0.385 +/- 0.418) telomere length decreased. These data suggest early activation of a telomere lengthening mechanism, prior to zygotic genome activation. Additionally, Intra-embryo telomere length increased until its peak day 3 (Coefficient of Variation = 108.51%) and was at its lowest at the blastocyst stage (Coefficient of Variation = 40.97%) when telomerase becomes active. Together these data suggest that an Alternative Lengthening of Telomeres (ALT) mechanism may be responsible for both increases in telomere length and increased heterogeneity among blastomeres within early embryos. Limitations, reasons for caution: The limited sample size for some of the earliest embryonic stages in addition to the freezing method for the embryos may have affected quality, and an inability to infer developmental ability as the embryos were not cultured to blastocyst. Wider implications of the findings: Our study is the first to perform a molecular characterization of telomere dynamics and the role of DNA damage in human oocytes and embryos at the single cell level and provides the first evidence that ALT is part of normal embryonic development in humans