SARA participation as a negative regulator of the TGFβ signaling pathway during neuronal development

VICTORIA ROZÉS SALVADOR; CECILIA CONDE; CARLOS WILSON; CHRISTIAN GONZALEZ BILLAULT

Chicago

Congreso; Neuroscience 2019; 2019

Society of Neuroscience

Several events are necessary for proper neuronal development, such as differential protein expression, cytoskeletal dynamics, and endosomal trafficking. Smad Anchor for Receptor Activation (SARA) is a protein that binds to early endosomes; carrying out specific functions related to traffic but also participating in signaling such as in Transforming Growth Factor β (TGFβ) pathway. In this regard, it has been described that SARA recruits Smad2/3 and, therefore, favors the activation of this pathway but also can modulate the TβRI dephosphorylation by PP1c, and inactivate the pathway both in epithelial cells and cell lines. Moreover, it has been shown that TGFβ signaling specifies axon during neuronal development; however, SARA participation in this signaling pathway during the developmental process remains unknown. For this reason we proposed to analyze the participation of SARA in TGFβ signaling during neuronal development. Results obtained in cultures of hippocampal neurons, through FRET Acceptor Photobleaching showed physical interaction between SARA and the TGFβ receptor I (TβRI). Also, performing loss and gain of function experiments, we found that SARA suppression (through shRNA expression) generates both greater axonal growth and loss of axonal specification since neurons have more than one axon compared with the control condition. In addition, the transition of polarity stage is accelerated in these neurons. Interestingly, this same phenotype is repeated when we use a mutant form of SARA (SARA-F728A) that alters its binding to PP1c protein (involved in TGFβ inactivation) and therefore, the TβRI remains hyperphosphorylated, keeping the pathway activated. Also, by FRET we found that SARA-F728A has more interaction with PP1c than control conditions, suggesting that SARA arrests to PP1c. Taking together; these results suggest that SARA participates in the TGFβ pathway in neurons through the negative regulation, which seems to be a necessary requirement for the correct neuronal development.