AbstractsBiology & Animal Science

The p75 neurotrophin receptor (p75NTR) is a multifunctional receptor that conveys several physiological functions such as cell death and survival, neurite outgrowth, differentiation, and cell migration, and it is also implicated in many pathological conditions. p75NTR belongs to the tumor necrosis factor superfamily (TNFRs) and as all members of this family, p75NTR lacks an intrinsic catalytic activity and the diverse signaling pathways it transduces are due to its interaction with different cytosolic adaptor proteins. Signaling of p75NTR through Rho GTPases has been described in many different processes such as death of oligodendrocytes, myelination, as well as inhibition of neurite outgrowth in response to myelin inhibitory proteins. However, the link between p75NTR and Rho GTPases activation is not well defined, mainly due to the lack of a robust assay that allows the study of p75NTR signaling. In this work we used the COS7 spreading assay as a heterologous system to study the regulation of Rho GTPases downstream of p75NTR. This assay is widely used as a model to study growth cone dynamics in response to semaphorin signaling. We find that p75NTR mediates COS7 cell spreading on laminin through activation of Rac1 and that cleavage of p75NTR by ADAM17 and γ-secretase is an essential step in this process. We have previously identified NRAGE, a MAGE family adaptor protein, as a p75NTR interactor through which p75NTR induces cell death. Here we show that NRAGE acts downstream of p75NTR to mediate Rac1 activation and cytoskeletal rearrangement. To gain more insight on how NRAGE mediates the spreading effects downstream of p75NTR we performed a yeast two-hybrid screen and identified NEDD9, a Cas family member as a potent NRAGE interactor. NEDD9 is widely studied as a prometastatic factor acting downstream of integrins and focal adhesion kinase. Here we show that NEDD9 acts downstream of p75NTR to mediate Rac1 activation and cell spreading. Methods available for the measurement of Rho GTPases activity do not take into account subtle changes in Rho GTPases activation and underestimate the importance of the spatiotemporal changes in their activity. In order to study how p75NTR regulates the activation of Rho GTPases we took advantage of the Rho biosensors that allow the measurement of Rho GTPases activity through FRET. Using this method we find that p75NTR constitutively activates Rac1 but does not change RhoA activity although results show a trend towards a decrease in RhoA activation. Importantly, we show that this balance is altered when coreceptors such as NogoR1 and myelin inhibitory proteins such as Nogo66 are present. p75NTR has been shown to play an important role in cell migration and cancer metastasis. In particular, p75NTR is a potent mediator of glioma invasion in vitro and in vivo. Here we show that p75NTR is overexpressed in high grade human glioma, and that p75NTR increases migration of U87MG cells in vitro using the agarose drop assay which is a 2D migration assay on laminin, as well as by following the…