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Annals of Medicine and Surgery 3 (2014) 18–19

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Annals of Medicine and Surgery

journal homepage: www.annals journal .com

Abstracts from the Barts and the London Academic Medicine and Surgery Society 2013 National ResearchConference on the 5th October 2013

Winning undergraduate oral presentationGLYCOGEN SYNTHASE KINASE 3b HAS A KINASE-INDEPENDENTFUNCTION IN NEURONAL MIGRATION DURING CORTICOGENESIS

Georgina Phillips, Tetsushi Kagawa, Tetsuya Taga.

Background: Disruption of neuronal migration during corticogenesis isimplicated in the development of many neurological and psychiatric dis-orders. Overexpression of Glycogen synthase kinase 3b (GSK3b) duringcorticogenesis inhibits neuronal migration. GSK3b is considered a prom-ising target in the development of new psychiatric therapies, althoughwhether it affects neuronal migration by kinase-dependent or indepen-dent mechanisms is unknown. This study aims to determine if GSK3b in-hibits neuronal migration through kinase-dependent mechanisms, using amouse model.Methods: Plasmids co-expressing green fluorescence protein with eitherGSK3b with no kinase activity “kinase dead” (GSK3bKD), constitutivelyactive GSK3b (GSK3bS9A), or no GSK3b (control) were transfected intoneural stem cells lining the lateral ventricles of embryonic day 13.5 C57BL6mice by in utero electroporation. Following two days of corticogenesis, themigratory patterns of developing neurons within the cortical plate (CP)were examined using fluorescence microscopy.Results: GSK3bKD transfected embryos had fewer neurons within the CPcompared to controls (p¼0.04) reflecting significantly reduced neuronalmigration. Similar inhibition of neuronal migration occurred withGSK3bS9A overexpression in the majority of mutants. Furthermore, asignificant reduction (p¼0.04) in unipolar neurons at the IntermediateZone-CP border in GSK3bKD mutants demonstrated cells’ failure to ach-ieve the polarity crucial for migration.Conclusions: These results provide the first evidence of a kinase-inde-pendent action of GSK3b in the inhibition of neuronal polarity and sub-sequent migration during corticogenesis, challenging the dogma of akinase-centric function of GSK3b. Further investigation, to determine po-tential kinase-independent mechanisms, could aid rational drug design forpsychiatric disorders.Acknowledgments: All Researchers at the Department of Stem CellRegulation, Tokyo Medical and Dental University, Japan.

Winning postgraduate oral presentationNG2/CSPG4 PROMOTES PROLIFERATION AND RESISTANCE TO THERAPYIN GLIOBLASTOMA MULTIFORME

Richard M. Heywood, Colin Watts.

Introduction: The NG2/CSPG4 proteoglycan has been shown to be amarker of an aggressive proliferative phenotype, and as a marker oftreatment resistant cells in glioblastoma (GBM). However, it is unknownwhether CSPG4 plays a functional role in these processes, and whetherthese in vitro findings have any clinical relevanceMethods: Freshly derived glioblastoma cells were used for all experi-ments. CSPG4 shRNA knockdown cells were assessed for proliferation,clonogenic potential and in vivo tumorigenicity. Immunocytochemicalstaining for EGFR and pEGFR, and pharmacological inhibition of EGFR,

were used to investigate interactions between CSPG4 and EGFR. Cells wereexposed to radiation, temozolomide and carmustine, and the proportion ofCSPG4+ cells was assessed by flow cytometry. Cell lines were FACS sorted,and the survival of sorted cells after treatment was asessed by growthcurve analysis. These experiments were repeated in CSPG4 knockdown celllines.Results: CSPG4 knockdown cells were less proliferative and clonogenicthan wild type cells. pEGFR staining was higher in wild type than inknockdown cells; wild type cells were more sensitive to pharmacologicalinhibition of EGFR. CSPG4 was enriched after exposure to all therapeuticmodalities. FACS sorting with functional assays confirmed that CSPG4+cells were more resistant to treatment. CSPG4 shRNA knockdown cellswere more sensitive to therapy.Conclusions: CSPG4 is a marker of cells that are more proliferative andmore resistant to the therapeutic modalities used clinically in the treat-ment of GBM. CSPG4 appears to have a functional role in these processes,by epigenetic stabilisation.Acknowledgments: Talal Al-Mayhani, Sara Piccirillo, Matt Rowland, SilviaSonzini and Anna Kolb.

Winning postersMIGRATION OF NEUTROPHILS IN TO LYMPHATICS STRUCTURESDURING INFLAMMATION IN VIVO: A ROLE FOR CCR7?

Jessica Ann Dilliway, Sussan Nourshargh, Mathieu-Benoit Voisin.

Background: Blood neutrophils are the first leukocytes to arrive at sites ofinfection and a new interest in neutrophil regulation of the adaptive im-mune responses has emerged over the past decade. However, very little isknown about the route of neutrophil migration into the lymphatic systemand this study aimed to evaluate the migration response of neutrophilsfrom the inflamed tissue, into the tissue-associated lymphatic vessels andfinally into the draining lymph nodes (LNs). Furthermore, the expression ofthe lymphatic chemokine receptor CCR7 on neutrophils was investigatedin vivo.Materials and Methods: WT mice received an intrascrotal injection ofTNFa or chick collagen II with Complete Freund's adjuvant. Cremastermuscles, proximal LNs, spleen and blood samples were harvested andthe presence of neutrophils in the different organs and expression ofCCR7 were quantified by confocal microscopy and flow cytometry,respectively.Results: Both TNFa and Collagen II+CFA induced migration of neutrophilsinto cremaster tissues but also into the cremasteric lymphatic vessels andproximal LNs. Extracellular staining of cells revealed very low levels ofsurface expression of CCR7. However, significant levels of intracellularstores of CCR7 could be detected.Conclusions: Our data indicates that both cytokine-induced and immu-nisation-based inflammation induces the migration of neutrophils into thetissue lymphatic vessels and proximal draining LNs. Furthermore, CCR7 isexpressed in mouse neutrophils mainly in intracellular stores. Collectively,these results indicate that CCR7 could play a role in the recruitment ofneutrophils into lymphatic structures upon models of cremastericinflammation in vivo.