(318a) Sensory Neurons Adapt Morphology to Environment Dimensionality by Modulation of Β1-Integrin Cytoskeleton Signaling: 3D Better Mimics In Vivo Features

Despite the great strides that have been made in developing new biomaterials, efforts to adapt these materials for nerve repair applications have been limited by a poor understanding of how neurons interact with their 3D environment. Thus, the ultimate goal of peripheral nerve tissue engineering is to provide rationally-designed alternatives to grafted tissue through a more comprehensive understanding of the dynamic 3D interactions between neurons and their environment. Neuronal signaling controls neurite outgrowth, and recent studies with non-neuronal cells demonstrated that signaling pathways are dramatically altered when cells are placed in a 3D matrix, with in vitro 3D environments being a better representation of in vivo systems compared to 2D models. We hypothesize that 3D culture imposes changes in matrix/ligand organization that affect neuronal behavior by modulating integrin signal transduction to the cytoskeleton. We cultured sensory neurons from mouse dorsal root ganglia (DRG) in 2D and 3D collagen gels. We examined and quantified the outgrowth of neuronal projections and the expression levels of Β1-integrin, RhoGTPases (Rac1 and RhoA), focal adhesion kinase (FAK) and the phosphorylation of FAK. Our work has established that DRG neuronal signaling pathways as well as neurite extension and branching are altered in 3D as a response to the recognition and adhesion of the neurons to their surrounding matrix. We note that 3D matrix adhesions form throughout the neurite length and within these adhesions, Β1-integrin expression is altered and FAK phosphorylation is dramatically decreased at Y397 suggesting neuronal independence of regulatory pathways involving FAK phosphorylation at this tyrosin site, which in 2D is mandatory for the regulation of neurite outgrowth. Thus a major conclusion of this work is that neuronal integrin signal transduction is significantly altered when cultured in 2D vs 3D presentations of the same substrate. This adaptation was manifest in growth cone morphology, neuronal polarity and neurite extension and branching, resulting in morphological features and neuronal maturation levels in 3D culture that more closely mimic those occurring in vivo. Taken together, our findings challenge the use of traditional 2D tissue culture conditions for understanding the in vivo structure of neurons and the signaling of neuronal adhesions in 3D microenvironments. Combined with the proper neural engineering tools, these results provide a strong foundation to design optimal biomaterials for the development of therapeutics for nerve repair and neurodegenerative disorders.