Cooperation between integrin and growth factor receptor signalling in the regulation of neurite growth from rat sensory neurons

Like growth and guidance of developing axons, regeneration after axonal injury is predominantly influenced by factors located in a neuron's extracellular environment. These factors include neurotrophins, such as Nerve Growth Factor (NGF), and adhesion molecules, such as laminin. However, how these factors interact to stimulate optimal levels of neurite growth is largely unknown. In this thesis, I initially investigated whether attachment to permissive culture substrates is sufficient to promote neurite outgrowth from DRG neurons, and whether this interaction is able to enhance the response of neurons to added neurotrophic factors, such as NGF. Adult Dorsal Root Ganglion (DRG) neurons plated on surfaces coated with a thin film of laminin exhibited increased neurite outgrowth, this growth was correlated with increased expression of laminin binding integrin subunits. Laminin-induced neurite growth was integrin-dependent as it was attenuated by treatment with either a cyclic Arg-Gly-Asp (RGD) peptide or a β1 integrin-blocking antibody (β1i). The addition of NGF to cells plated on laminin resulted in a significant and synergistic increase in the integrin-dependent outgrowth. -- In performing these experiments it was observed that not all DRG neurons were responding to laminin and NGF with increased neurite growth. As neurons in the adult rat DRG can be classified into at least three separate subpopulations based on morphologic and phenotypic differences, a cell selection approach was utilized to show that laminin-induced neurite growth occurs in the absence of added trophic factors only in heavy chain neurofilament positive and calcitonin gene related peptide positive DRG neurons (NGF-responsive population). In contrast, laminin alone is not sufficient to stimulate significant neurite growth from lectin Griffonia simplicifolia 1B4 positive neurons (IB4+ve), although it is still required to elicit a growth response from these cells in the presence of Glial Cell Line Derived Neurotrophic Factor (GDNF) (e.g., neurite growth occurred only when cells were plated on laminin in the presence of GDNF). By using chemical inhibitors we demonstrated that only the PI3-K/Akt pathway was required for neurite growth from the NGF-responsive cell population. However, both the PI3-K/Akt and MEK/MAPK signalling pathways were required for neurite growth from the IB4+ve cell population. Further analysis indicated that Src was potentially a key point of collaboration between NGF and laminin induced neurite growth in NGF-responsive adult DRG neurons. Src appeared to be located upstream of the PI3-K/Akt signalling pathway and was responsible for increased neurite growth induced by the addition of laminin and NGF together. -- In this series of studies we have identified specific signalling events and environmental requirements associated with neurite growth for different subpopulations of adult DRG neurons, pointing to potential therapeutic targets while identifying why any one treatment alone maybe insufficient to totally repair peripheral nerve damage