Browsing by Author "Neves, Ana Catarina Gomes das"
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- The role of microtubules in bouton initiation and outgrowthPublication . Neves, Ana Catarina Gomes das; Teodoro, Rita; Marques-Ramos, AnaABSTRACT: Plasticity is the process through which neurons adapt their shape and function to external stimuli and is a critical process for development, learning, and memory. New findings indicate that the formation of new and immature synaptic structures – ghost boutons (GBs) – in motor neurons of Drosophila melanogaster (D. melanogaster) occurs through a mechanism that has never been reported in neuron remodeling, called blebbing, in response to acute stimuli that replicate conditions of high synaptic activity. It was shown that actin does not contribute to the initiation and outgrowth of new synaptic boutons by blebbing, being actin required in later stages of development like stabilization and maturation of GBs. However, the specific role of other cytoskeleton elements, such as microtubules (MTs), in this process remains poorly understood. Thus, this study aims to characterize the dynamics of MTs in the formation and growth of GBs in D. melanogaster. This study employed genetic manipulation techniques, including knockdown and overexpression of microtubule-binding proteins, and immunohistochemical staining for α- and β-tubulin. Third-instar D. melanogaster larvae were stimulated with a patterned high-potassium and calcium protocol to induce the formation of additional GBs. MT dynamics were further probed using taxol, a stabilizing agent, and vinblastine, a destabilizer, to manipulate MT stability during GB development. The labeling of α- and β-tubulin revealed dynamic changes in MT stability within GBs. Supporting this, taxol treatment led to a significant reduction in GB numbers, yet the remaining GBs were larger and often contained high intensity of the stabilizing protein Futsch, suggesting that MT stabilization may inhibit initial GB formation but promote the growth of mature boutons. Conversely, vinblastine treatment did not significantly alter GB numbers, indicating that complete MT destabilization may interfere with the maturation process, suggesting that a degree of MT stabilization may be required for GB maturation. EB1, a plus-end tracking protein, was present in all GBs possibly indicating its involvement in the initiation of the blebbing process and that MT polymerization may be happening during GB development. Futsch and Short-Stop proteins, which help stabilize MTs, exhibited similar results regarding their presence within GBs and decrease in GB numbers when their expression is reduced, highlighting their roles in promoting GB stability and MT integrity, indicating that they are crucial for the blebbing process. Lastly, the severing protein subunit Katanin-60, played a critical role in bouton development, as reduced levels of Katanin-60 resulted in a decrease in GB numbers, highlighting the importance of controlled MT severing for proper GB formation and maintenance. Thus, our findings highlight the crucial role of MT dynamics in GB formation, suggesting that both MT stabilizers and severing proteins are involved in synaptic plasticity. These findings might provide relevant insights into potential therapeutic approaches for neurodegenerative diseases linked to synaptic dysfunction, such as Amyotrophic Lateral Sclerosis and Alzheimer’s disease. Future studies, including live imaging, behavioral analysis, and Tau-Spastin compensatory mechanism assessment, are needed to elucidate the exact processes underlying MT involvement in GB formation and outgrowth.