| dc.description.abstracteng | In many traumatic and neurodegenerative disorders of the central nervous system, axonal degeneration is a central and early pathophysiological feature. Since the regenerative response of injured neurons is severely hampered by an intrinsically reduced growth capacity and external growth-inhibiting factors such as CSPG, axonal degeneration often results in progressive clinical disability. An improved understanding of the molecular mechanisms is hoped to unravel potential therapeutic targets, as there are currently no therapeutic options available. During axonal degeneration, macroautophagy (here: autophagy), a cellular homeostatic process responsible for the degradation of long-lived organelles and proteins, was previously shown to be activated, but its detailed role in this context, harmful or beneficial, is incompletely understood. Particularly, the autophagy-initiating kinase ULK1 accumulated in degenerating axons, leading to the speculation that ULK1-dependent autophagy might represent a deleterious executing process during axonal degeneration. To assess this hypothesis in the present study, primary cortical neurons were transduced with an adeno-associated viral vector expressing kinase-dead dominant-negative ULK1, leading to the inhibition of ULK1 function. After selective axonal lesion to transduced neurons cultured in microfluidic chambers, dominant-negative ULK1 attenuated acute axonal degeneration for up to six hours and fostered axonal regeneration up to 96 hours post-injury, as compared with control. Correspondingly, increased neurite outgrowth was observed after transduction with dominant-negative ULK1 in neurons cultured on both the permissive substrate laminin and the growth-inhibiting matrix CSPG. Mechanistically, dominant-negative ULK1 reduced autophagy activation, indicating that a decrease in autophagy is one mechanism underlying its axon-protective effects. The additional proteomic analysis of neurons transduced with dominant-negative ULK1 surprisingly outlined a strong regulation of splicing and translation-associated proteins. This finding was corroborated by a transcriptomic analysis uncovering dominant-negative ULK1-dependent differential splicing of axonal degeneration and regeneration-associated genes such as Kif1b and Ddit3. Dominant-negative ULK1 might therefore additionally elicit axon-protective and pro-regenerative properties via a modulation of Kif1b-dependent axonal transport and Ddit3-mediated endoplasmic reticulum stress. Furthermore, the beneficial effects of dominant-negative ULK1 on axonal degeneration and regeneration were connected to an activation of mTOR-S6-mediated translation, elevated expression of the growth-promoting molecule p-ERK1, and reduced levels of the degeneration-mediator and growth-inhibitor ROCK2. Together, the data obtained in this thesis reveal a key involvement of ULK1 in axonal degeneration and regeneration in vitro, and demonstrate a more complex role of ULK1 in neuronal biology than previously known, including a novel function in splicing. ULK1 inhibition attenuates the degeneration of axons and promotes their regeneration in vitro by a proposed switch from the catabolic autophagy process to the activation of axon-protective and axon growth-promoting processes. ULK1 inhibition thus represents a putative therapeutic approach in traumatic and neurodegenerative disorders of the central nervous system. | de |