Neurons are the most morphologically complex cell type in multicellular organisms, and this complexity is intricately linked to the complexity of nervous system information processing. Thus, impairments in neuronal morphogenesis invariably result in alterations of nervous system function and often debilitating neurological disease. Precise control of microtubule cytoskeleton organization is central to most if not all aspects of neuron development and function ranging from the migration of newborn neurons, cycles of neurite elongation, retraction and branching to growth cone guidance and synapse formation. This importance of neuronal microtubules is reflected in the wide range of neurodevelopmental and neurodegenerative diseases linked to genetic defects in proteins associated with the microtubule cytoskeleton. Human genetics and modern sequencing techniques identified numerous mutations in related genes associated with frequently severe cortical malformations. These include both mutations of tubulin genes themselves ? so-named tubulinopathies ? as well as mutations in neuronal microtubule-associated proteins that often have a similar range of neurodevelopmental phenotypes. While it is generally assumed that these mutations disrupt the developmental migration of immature neurons through the developing cortex, we do not understand the rules governing MT function in neuromorphogenesis at a mechanistic level. In this application, we propose to use emerging human induced pluripotent stem cell (iPSC) technology in combination with state-of-the-art genome engineering and our established expertise in quantitative microscopy to model and dissect the neuronal cell biology of tubulinopathy-like diseases and the function of dynamic MTs in neuromorphogenesis.
In Aim 1, we focus on DCX and tubulin mutations that cause cortical malformations, and we will analyze how DCX controls neuronal microtubule dynamics and mechanics, and neuronal morphogenesis based on our recent data that DCX binds microtubules in a unique geometry-dependent way.
In Aim 2, we employ novel optogenetics to control protein interactions with growing microtubule plus ends with second and micrometer precision to map how microtubule plus end complexes contribute to neuronal development dynamics. We believe that quantitative and rigorous understanding of the principles that govern MT function in neuronal development and what goes wrong in neurodevelopmental disease will have tangible and important outcomes for human health, and can lay the foundation for future therapeutic approaches.
Mutations in tubulin, a protein that assembles into a dynamic intracellular microtubule filament system, and in microtubule-associated proteins such as doublecortin result in a spectrum of brain malformations in which developing neurons do not reach their proper destination in the developing brain. Based on our recent findings that doublecortin binds to microtubules in a unique way, we dissect the molecular mechanisms by which doublecortin contributes to neuronal development using a novel human neuronal differentiation model, and utilize innovative optogenetics to spatially and temporally dissect the rules by which microtubule dynamics control neuronal development. This research contributes to our fundamental understanding of nervous system development and its impairment in disease.
