Understanding how GSK3b controls neuronal development in time and space

How extracellular stimuli control and maintain neurons is a fundamental question, with important implications for treatment of neuronal diseases. Microtubule (MT) function is critical for neurodevelopment and dysregulation leads to neurodegenerative diseases, neuropathies and impaired regeneration after injury. GSK-3ß is a key coordinator of MT regulation. It is known to ameliorate various neurological conditions, but in the short term and with off-target effects (e.g. Lithium). This important molecule is therefore a major focus of research and a promising therapeutic target. GSK-3ß is found in all cellular compartments, but global inhibition has not been therapeutically beneficial. There are major controversies over whether GSK-3ß promotes or inhibits neuronal outgrowth and by which mechanism this would be mediated. We believe that GSK-3ß's remarkable specificity and range of functions are possible because of its dynamic activity patterns in time and space. To investigate this, we will develop novel GSK-3ß activity biosensors and locally targeted GSK-3ß variants to explore how these spatio-temporal dynamics coordinate neuronal development and maintenance. Our team is uniquely positioned to undertake this research: Ines Hahn has developed a Drosophila neuron model to study GSK-3ß and its target protein networks, Peter O’Toole is an expert using cutting-edge imaging equipment and FRET approaches and Han-Jou Chen studies neurodegeneration in rodent cell cultures. Together with our project partner Klaus Hahn (UNC/USA), a world-leading expert in biosensor generation, we will pursue two primary objectives: Objective 1) Visualise GSK-3ß activity in living fly and rat neurons by pioneering the development of GSK-3ß FRET biosensors that will enable us to measure GSK-3ß activity live for the first time. We will use two independent sensor approaches that have strong proof of concept data and complement them with a systematic analysis of in/active GSK-3ß pools using established antibodies as a third lower-risk approach. Objective 2) Identify the GSK-3ß target proteins that drive the local changes in microtubule dynamics: We will investigate whether GSK-3ß activity pulses and/or regions of active GSK-3ß coincide with changes in microtubule dynamics. Using in/active GSK-3ß variants and peptide inhibitors localised to specific cellular regions we will test whether localized activation of GSK-3ß promotes mcirotubule unbundling and reduction activity triggers stabilisation events. We will then examine if those effects are suppressed in mutants of key microtubule binding proteins we identified previously. Despite over three decades of research, specifics of GSK-3ß’s function remain contradictory. We believe that quantifying its activity in neurons will provide new insights into its role. Understanding how localized kinase activity translates into coordinated neuronal function could lead to innovative therapeutic strategies, such as targeting specific subcellular GSK-3ß pools through our locally restricted inhibitors. Those approaches hold promise for addressing cognitive decline, neurodegenerative and developmental diseases, which are significant societal challenges; the cost of dementia is projected to triple by 2040, underscoring the importance of this work within BBSRC’s Major Challenge of “Understanding the rules of life” and ‘securing better health, ageing, and wellbeing.’ Drosophila offers a unique model for studying complex signalling processes, enabling rapid functional data acquisition while minimizing the use of higher-order animals. This work has potential impacts benefiting those studying neuronal function, injury, survival, and neurodevelopment and -degeneration. Our novel tools will also advance GSK-3ß biology in other areas, including cell division, cancer, inflammation, cardiovascular biology, and metabolism, contributing to significant advancements in other BBSRC target areas.