Abstract (eng)
During neuronal development axons respond to an array of signals to navigate to their targets. Growth cones at the tip of the axons choose their path with the aid of extracellular guidance cues. They are composed of finger like projections, filopodia and veil like structure called lamellipodia. Cues can either attract or repel growth cones. Extracellular cues are the molecules that are secreted or presented on the cell surface by cells along the axon path including netrin, semaphorins, slits and ephrins. These guidance cues regulate growth cone advance, turning, and branching behaviours of the growth through cytoskeleton rearrangements.
Growth cone dynamics is regulated by two cytoskeleton components, actins and microtubules (MTs). MTs are extremely dynamic structures, involved in neurite extension, retraction and polarity. The dynamics of microtubule is regulated by Microtubule associated protein 1B (MAP1B), a class of proteins which bind along the MTs and regulate their function in growth and guidance. MAP1B KO display various defects including agenesis of corpus callosum, misguided commisural forming probst bundles.
Recently known guidance cue draxin is an important repulsive axon guidance cue essential for the formation of spinal cord and forebrain commissures, including corpus callosum. Draxin is expressed in various brain regions including, cortex, olfactory bulb, midbrain and cerebellum. It inhibits the neurite outgrowth from dorsal spinal cord, olfactory bulb and cortical explants in vitro. Draxin induces neurite outgrowth inhibition through multiple netrin receptors: DCC (deleted in colorectal cancer), Neogenin, UNC5s (H1, H2, H3), and DSCAM (Down's syndrome cell adhesion molecule) but the molecular details of draxin signaling are unknown.
In the first part of my thesis I examined the involvement of MAP1B in draxin and Semaphorin3A (Sema3A) induced neurite outgrowth inhibition and growth cone collapse. In order to address these questions, I established two assays. For neurite out growth assay cerebral cortical explants were used whereas for growth cone collapse assay dissociated cortical neurons were used.
These assays suggested that draxin and Sema3A signaling is dependent on MAP1B. Using genetic and pharmacological approaches I found that draxin-induced growth cone collapse critically depends on draxin receptors (deleted in colorectal cancer, DCC), inhibition of protein kinase Akt and activation of GSK-3β (glycogen synthase kinase-3β) which correlates with increased phosphorylation of MAP1B. This study, for the first time reveals molecular mechanisms involved in draxin repulsion, links draxin and DCC to MAP1B and identifies a novel MAP1B-depenent GSK-3β pathway essential for repulsive axon guidance. Additionally, I studied the effect of draxin on polarization of dissociated cortical neurons and the effect of Sema3A on branching of pyramidal neurons.
In the second part of my study I investigated the role of myosin in nitric oxide induced axon retraction in mouse neuroblastoma (N2a) cell lines. Myosin is motor protein involved in the acto-myosin contractility. Using biochemical assay I analyzed the monophosphorylation of the myosin regulatory light chain (MRLC) at Ser19 as an indicator of the myosin activity. N2a cells were treated with SNAP, a Nitric oxide (NO) donor. Treatment of SNAP enhanced the monophosphorylation of the MRLC. The increase in phosphorylation was partially inhibited by the rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitor Y27632, suggesting that ROCK is the key to myosin activation in response to SNAP. These results demonstrated that myosin is important for the axon retraction induced by NO.