Faculty Bibliography

The COP9 signalosome removes Nedd8 modifications from the Cullin subunits of ubiquitin ligase complexes, reducing their activity. Here we show that mutations in the Drosophila COP9 signalosome subunit 1b (CSN1b) gene increase the activity of ubiquitin ligases that contain Cullin 1. Analysis of CSN1b mutant phenotypes revealed a requirement for the COP9 signalosome to prevent ectopic expression of Epidermal growth factor receptor (EGFR) target genes. It does so by protecting Capicua, a transcriptional repressor of EGFR target genes, from EGFR pathway-dependent ubiquitination by a Cullin 1/SKP1-related A/Archipelago E3 ligase and subsequent proteasomal degradation. The CSN1b subunit also maintains basal Capicua levels by protecting it from a separate mechanism of degradation that is independent of EGFR signaling. As a suppressor of tumor growth and metastasis, Capicua may be an important target of the COP9 signalosome in cancer.

Chronic rejection significantly limits long-term success of solid organ transplantation. De novo donor-specific antibodies (DSAs) to mismatched donor human leukocyte antigen after human lung transplantation predispose lung grafts to chronic rejection. We sought to delineate mediators and mechanisms of DSA pathogenesis and to define early inflammatory events that trigger chronic rejection in lung transplant recipients and obliterative airway disease, a correlate of human chronic rejection, in mouse. Induction of transcription factor zinc finger and BTB domain containing protein 7a (Zbtb7a) was an early response critical in the DSA-induced chronic rejection. A cohort of human lung transplant recipients who developed DSA and chronic rejection demonstrated greater Zbtb7a expression long before clinical diagnosis of chronic rejection compared to nonrejecting lung transplant recipients with stable pulmonary function. Expression of DSA-induced Zbtb7a was restricted to alveolar macrophages (AMs), and selective disruption of Zbtb7a in AMs resulted in less bronchiolar occlusion, low immune responses to lung-restricted self-antigens, and high protection from chronic rejection in mice. Additionally, in an allogeneic cell transfer protocol, antigen presentation by AMs was Zbtb7a-dependent where AMs deficient in Zbtb7a failed to induce antibody and T cell responses. Collectively, we demonstrate that AMs play an essential role in antibody-induced pathogenesis of chronic rejection by regulating early inflammation and lung-restricted humoral and cellular autoimmunity.

BACKGROUND: The number of hip fractures is rising as life expectancy increases. As such, the number of centenarians sustaining these fractures is also increasing. The purpose of this study was to determine whether patients who are >/=100 years old and sustain a hip fracture fare worse in the hospital than those who are younger. METHODS: Using a large database, the New York Statewide Planning and Research Cooperative System (SPARCS), we identified patients who were >/=65 years old and had been treated for a hip fracture over a 12-year period. Data on demographics, comorbidities, and treatment were collected. Three cohorts were established: patients who were 65 to 80 years old, 81 to 99 years old, and >/=100 years old (centenarians). Outcome measures included hospital length of stay, estimated total costs, and in-hospital mortality rates. RESULTS: A total of 168,087 patients with a hip fracture were identified, and 1,150 (0.7%) of them had sustained the fracture when they were >/=100 years old. Centenarians incurred costs and had lengths of stay that were similar to those of younger patients. Despite the similarities, centenarians were found to have a significantly higher in-hospital mortality rate than the younger populations (7.4% compared with 4.4% for those 81 to 99 years old and 2.6% for those 65 to 80 years old; p < 0.01). Male sex and an increasing number of medical comorbidities were found to predict in-hospital mortality for centenarians sustaining extracapsular hip fractures. No significant predictors of in-hospital mortality were identified for centenarians who sustained femoral neck fractures. An increased time to surgery did not influence the odds of in-hospital mortality. CONCLUSIONS: Centenarians had increased in-hospital mortality, but the remaining short-term outcomes were comparable with those for the younger cohorts with similar fracture patterns. For this extremely elderly population, time to surgery does not appear to affect short-term mortality rates, suggesting a potential benefit to preoperative optimization. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Endothelial cell adhesion and migration are critical steps of the angiogenic process, whose dysfunction is associated with tumor growth and metastasis. The TRPM8 channel has recently been proposed to play a protective role in prostate cancer by impairing cell motility. However, the mechanisms by which it could influence vascular behavior are unknown. Here, we reveal a novel non-channel function for TRPM8 that unexpectedly acts as a Rap1 GTPase inhibitor, thereby inhibiting endothelial cell motility, independently of pore function. TRPM8 retains Rap1 intracellularly through direct protein-protein interaction, thus preventing its cytoplasm-plasma membrane trafficking. In turn, this mechanism impairs the activation of a major inside-out signaling pathway that triggers the conformational activation of integrin and, consequently, cell adhesion, migration, in vitro endothelial tube formation, and spheroid sprouting. Our results bring to light a novel, pore-independent molecular mechanism by which endogenous TRPM8 expression inhibits Rap1 GTPase and thus plays a critical role in the behavior of vascular endothelial cells by inhibiting migration.

Tissue homeostasis of skin is sustained by epidermal progenitor cells localized within the basal layer of the skin epithelium. Post-translational modification of the proteome, such as protein phosphorylation, plays a fundamental role in the regulation of stemness and differentiation of somatic stem cells. However, it remains unclear how phosphoproteomic changes occur and contribute to epidermal differentiation. In this study, we survey the epidermal cell differentiation in a systematic manner by combining quantitative phosphoproteomics with mammalian kinome cDNA library screen. This approach identified a key signaling event, phosphorylation of a desmosome component, PKP1 (plakophilin-1) by RIPK4 (receptor-interacting serine-threonine kinase 4) during epidermal differentiation. With genome-editing and mouse genetics approach, we show that loss of function of either Pkp1 or Ripk4 impairs skin differentiation and enhances epidermal carcinogenesis in vivo Phosphorylation of PKP1's N-terminal domain by RIPK4 is essential for their role in epidermal differentiation. Taken together, our study presents a global view of phosphoproteomic changes that occur during epidermal differentiation, and identifies RIPK-PKP1 signaling as novel axis involved in skin stratification and tumorigenesis.

The ovaries have a fixed pool of immature (primordial) follicles at birth known as the ovarian reserve. Activation or loss of follicles in this reserve causes an irreversible decline in reproductive function that culminates in menopause. Premenopausal women with cancer who are treated with conventional genotoxic chemotherapy have accelerated loss of the ovarian reserve, leading to subfertility and infertility. Cyclophosphamide (CY), a highly gonadotoxic drug, is widely used as part of combination cancer chemotherapy. This drug induces ovarian damage in large part by activating the mammalian/mechanistic target of rapamycin (mTOR) pathway, leading to activation of primordial follicles, follicular burnout, and premature menopause. As a result, the probability of pregnancy in premenopausal female cancer survivors is significantly diminished. There has been little progress in preserving ovarian function during cancer chemotherapy. As part of multiagent chemotherapeutic regimens, inhibitors of themTORC1 pathway have a growing role in cancer treatment and are being studied as treatment for a growing number of malignant and nonmalignant conditions. Mammalian/mechanistic target of rapamycin inhibitors block the primordial-to-primary follicle transition. The investigators used a clinically relevant mouse model of chemotherapy-induced gonadotoxicity to investigate whether inhibitors of mTOR could block CY-induced premature activation of primordial follicles and also preserve fertility during chemotherapy. Two inhibitors of the mTOR pathway were used: everolimus (RAD001), a drug clinically approved for treatment of tamoxifen-resistant or relapsing estrogen receptor-positive breast cancer), and INK128, an experimental drug. Female mice were treated with CY weekly and then randomized to also receive either everolimus, INK128, or nothing.

BACKGROUND:Dalbavancin is a novel lipoglycopeptide antibiotic that has potent in vitro activity against Gram-positive microorganisms. METHODS:We performed a phase 1, open-label, multicenter study to investigate the pharmacokinetics (PK) and safety of a single dose of intravenous dalbavancin in hospitalized pediatric subjects 3 months to 11 years of age. We combined these data with previously collected adolescent PK data and performed a population PK analysis. RESULTS:Model development was performed using 311 dalbavancin plasma concentrations from 43 subjects. The median age was 5.9 years (range: 0.3-16.9). A 3-compartment, linear PK model was developed. Based on simulations, the following age-dependent dosing regimen was found to achieve similar dalbavancin exposure to that in adults administered a 2-dose regimen: children 6 to <18 years of age, 12 mg/kg (1000 mg maximum) on day 1 and 6 mg/kg (500 mg maximum) on day 8 and children 3 months to <6 years of age, 15 mg/kg (1000 mg maximum) on day 1 and 7.5 mg/kg (500 mg maximum) on day 8. Similarly, the following age-dependent regimen was found to match adult exposure after a single-dose (1500 mg): 6 to <18 years of age, 18 mg/kg (1500 mg maximum) on day 1 and 3 months to <6 years of age, 22.5 mg/kg (1500 mg maximum) on day 1. Nineteen subjects experienced 36 treatment-emergent adverse events. Five of 36 adverse events were assessed as possibly or probably related to treatment. CONCLUSIONS:Dalbavancin pediatric dosing that matched adult exposure was identified. Overall, dalbavancin was well tolerated in our study population.

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

We examined the usefulness of commercially available DNA methylation arrays designed for the human genome (Illumina HumanMethylation450 and MethylationEPIC) for high-throughput epigenome analysis of the common marmoset, a nonhuman primate suitable for research on neuropsychiatric disorders. From among the probes on the methylation arrays, we selected those available for the common marmoset. DNA methylation data were obtained from genomic DNA extracted from the frontal cortex and blood samples of adult common marmosets as well as the frontal cortex of neonatal marmosets. About 10% of the probes on the arrays were estimated to be useful for DNA methylation assay in the common marmoset. Strong correlations existed between human and marmoset DNA methylation data. Illumina methylation arrays are useful for epigenome research using the common marmoset.

Recent data suggest that intraneuronal accumulation of metabolites of the amyloid-beta-precursor protein (APP) is neurotoxic. We observed that transgenic mice overexpressing in neurons a human APP gene harboring the APPE693Q (Dutch) mutation have intraneuronal lysosomal accumulation of APP carboxylterminal fragments (APP-CTFs) and oligomeric amyloid beta (oAbeta) but no histological evidence of amyloid deposition. Morphometric quantification using the lysosomal marker protein 2 (LAMP-2) immunolabeling showed higher neuronal lysosomal counts in brain of 12-months-old APPE693Q as compared with age-matched non-transgenic littermates, and western blots showed increased lysosomal proteins including LAMP-2, cathepsin D and LC3. At 24 months of age, these mice also exhibited an accumulation of alpha-synuclein in the brain, along with increased conversion of LC3-I to LC3-II, an autophagosomal/autolysosomal marker. In addition to lysosomal changes at 12 months of age, these mice developed cholinergic neuronal loss in the basal forebrain, GABAergic neuronal loss in the cortex, hippocampus and basal forebrain and gliosis and microgliosis in the hippocampus. These findings suggest a role for the intraneuronal accumulation of oAbeta and APP-CTFs and resultant lysosomal pathology at early stages of Alzheimer's disease-related pathology.Molecular Psychiatry advance online publication, 25 October 2016; doi:10.1038/mp.2016.189.