Faculty Bibliography

Regulatory T cells (Tregs) are an essential component of the immune system, but are also involved in the suppression of anti-tumour immune responses. The study examines their immunoregulatory role including their transcription factor, FOXP3, in oral and cutaneous SCC. Tregs were detected by double-immunohistochemistry. FOXP3-mRNA-expression was examined in tumour tissue, as well as in skin-derived primary cells and cell lines of different malignancy. Tregs were found in the tumour microenvironment, and FOXP3-mRNA-expression was significantly higher than in normal skin. Intriguingly, single FOXP3+ cells exhibited morphologic characteristics of SCC cells. Consistent with this, endogenous FOXP3-mRNA-expression was indeed detected in the epidermal cell lineage and dramatically increased with increasing malignancy of the cells. SCCs recruit Tregs into their microenvironment, presumably in order to suppress immunosurveillance, thus avoiding destruction by the immune system. Endogenous FOXP3-expression in malignant epidermoid cells might present a novel mechanism of immune escape.

The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes. The mechanisms by which these progenitors are able to expand within the PM microenvironment without premature differentiation remain largely unknown. Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM. CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM. Notably, the phenotype of N-cadherin deficiency could be partially rescued by activating Wnt signaling, suggesting a delicate functional interaction between the adhesion role of N-cadherin and Wnt signaling in the early PM microenvironment. This study suggests a new mechanism for the early renewal of AHF progenitors where N-cadherin provides additional adhesion for progenitor cells in the PM, thereby allowing Wnt paracrine signals to expand the cells without premature differentiation.Cell Research advance online publication 4 November 2014; doi:10.1038/cr.2014.142.

The capability to conditionally inactivate gene function is essential for understanding the molecular basis of development. In gene and mRNA targeting approaches, protein products can perdure, complicating genetic analysis. Current methods for selective protein degradation require drug treatment or take hours for protein removal, limiting their utility in studying rapid developmental processes in vivo. Here, we repurpose an endogenous protein degradation system to rapidly remove targeted C. elegans proteins. We show that upon expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, proteins tagged with the ZF1 zinc-finger domain can be quickly degraded in all somatic cell types examined with temporal and spatial control. We demonstrate that genes can be engineered to become conditional loss-of-function alleles by introducing sequences encoding the ZF1 tag into endogenous loci. Finally, we use ZF1 tagging to establish the site of cdc-42 gene function during a cell invasion event. ZF1 tagging provides a powerful new tool for the analysis of dynamic developmental events.

The etiology of endometriosis remains poorly understood but circulating stem cells may contribute. Telomeres shorten with cell divisions and age. Stem cells attempt to compensate for telomere attrition through the action of telomerase. Since circulating stem cells may contribute to endometriosis, we compared telomere content in lymphocytes of patients with and without endometriosis. METHODS: Observational study comparing peripheral lymphocytes telomere content, measured by quantitative polymerase chain reaction, in patients with (n = 86) and without endometriosis (n = 21). FINDINGS: Patients with endometriosis had longer telomeres than that of matched, endometriosis-free controls (telomere to single copy gene ratio [T/S ratio] of 1.62 vs 1.34, respectively, P = .00002). Patients with endometriosis were 8.1-fold more likely to have long telomeres. (odds ratio = 8.1, 95% confidence interval: 1.28-51.57, P = .0264). INTERPRETATION: Longer telomeres could be consistent with a stem cell origin of endometriosis.

Melanocortin receptor four (MC4R) is implicated in regulation of stress-related functions. We previously demonstrated that intranasal infusion of MC4R antagonist HS014, shortly before single prolonged stress (SPS) animal model of post-traumatic stress disorder, lessened the development of anxiety- and depression-like behavior depending on the dose. Here, we evaluated effects of HS014 on SPS-elicited changes in hypothalamic-pituitary-adrenal axis and expression of several genes of interest in mediobasal hypothalamus, hippocampus, and locus coeruleus. Rats were given intranasal infusion of HS014 (3.5 ng or 100 mug) and 30 min later subjected to SPS stressors. Short-term responses of HS014 rats in comparison with vehicle-treated, evident 30 min following SPS stressors, included smaller rise in plasma corticosterone (100 mug HS014), absence of induction of corticotrophin-releasing hormone mRNA in mediobasal hypothalamus and of mRNA for tyrosine hydroxylase and dopamine-beta hydroxylase in locus coeruleus. Long-term responses found 7 days after SPS stressors, included lower induction corticotrophin-releasing hormone mRNA levels in the mediobasal hypothalamus without effect on mRNAs for the glucocorticoid receptor (GR) and FK506-binding protein 51 (FKBP5), a component of GR co-chaperone complex; and no induction of GR protein in ventral hippocampus. Thus, antagonism of MC4R prior to SPS attenuates development of several abnormalities in gene expression in regions implicated in post-traumatic stress disorder. Blockade of brain melanocortine receptor 4 (MC4R) with intranasal infusion of the MC4R antagonist HS014 to rats prior to single prolonged stress (SPS) leads to faster termination of stress responses (30 min later) and prevents or attenuates SPS-triggered abnormal gene expression related to post-traumatic stress disorder (7 days later). Targeting of brain MC4R is a promising strategy to protect HPA axis, LC-NE (locus coeruleus-norepinephrine) systems and hippocampus from overstimulation.

Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimer's disease and Alzheimer mouse models. We have previously reported that severe lysosomal and amyloid neuropathology and associated cognitive deficits in the TgCRND8 Alzheimer mouse model can be ameliorated by restoring lysosomal proteolytic capacity and autophagy flux via genetic deletion of the lysosomal protease inhibitor, cystatin B. Here we present evidence that macroautophagy is a significant pathway for lipid turnover, which is defective in TgCRND8 brain where lipids accumulate as membranous structures and lipid droplets within giant neuronal autolysosomes. Levels of multiple lipid species including several sphingolipids (ceramide, ganglioside GM3, GM2, GM1, GD3 and GD1a), cardiolipin, cholesterol and cholesteryl esters are elevated in autophagic vacuole fractions and lysosomes isolated from TgCRND8 brain. Lipids are localized in autophagosomes and autolysosomes by double immunofluorescence analyses in wild-type mice and colocalization is increased in TgCRND8 mice where abnormally abundant GM2 ganglioside-positive granules are detected in neuronal lysosomes. Cystatin B deletion in TgCRND8 significantly reduces the number of GM2-positive granules and lowers the levels of GM2 and GM3 in lysosomes, decreases lipofuscin-related autofluorescence, and eliminates giant lipid-containing autolysosomes while increasing numbers of normal-sized autolysosomes/lysosomes with reduced content of undigested components. These findings have identified macroautophagy as a previously unappreciated route for delivering membrane lipids to lysosomes for turnover, a function that has so far been considered to be mediated exclusively through the endocytic pathway, and revealed that autophagic-lysosomal dysfunction in TgCRND8 brain impedes lysosomal turnover of lipids as well as proteins. The amelioration of lipid accumulation in TgCRND8 by removing cystatin B inhibition on lysosomal proteases suggests that enhancing lysosomal proteolysis improves the overall environment of the lysosome and its clearance functions, which may be possibly relevant to a broader range of lysosomal disorders beyond Alzheimer's disease.