Building a brain : interrogating how the α-tubulin gene TUBA1A contributes to neurodevelopment

Abstract

The importance of microtubules during the development and appropriate function of the nervous system has long been acknowledged. The highly complex coordination of cell proliferation, differentiation, migration, neuronal outgrowth and polarization, and synapse formation relies on the proper regulation of microtubules, which are dynamic cytoskeletal polymers that help provide structure and generate force in all eukaryotic cell types. However, how microtubules are regulated to support distinct, context-specific roles in different compartments of the neuron and at different stages of development is widely unknown. Recently, the identification of diverse neurodevelopmental defects associated with mutations to tubulin genes supports the hypothesis that neurons are particularly sensitive to disruptions to their microtubule network. Further, the wide array of patient phenotypes suggest that mutations to different tubulin isotypes, and even distinct variants within the same tubulin gene, can lead to drastically different outcomes during brain development. This suggests that mutations to tubulin genes disrupt distinct molecular functions, and that these different molecular “tweaks” to the microtubule network can lead to different, large-scale developmental consequences observed in patients. Interrogating the molecular consequences of these patient-derived tubulin mutations will provide insight not only into the disease progression of devastating tubulinopathies, but also shed light on specific requirements of the microtubule network during neurodevelopment. In this dissertation, I will discuss the important role of the primary, neuronally-expressed α-tubulin isotype, TUBA1A, in neurodevelopment. Dominant, de novo mutations to TUBA1A are associated with an array of cortical malformations, intellectual deficits, and frequently epilepsy and paralysis. I investigate the molecular, cellular, and tissue-level consequences of the most common variants to TUBA1A identified in patients. These mutations alter a conserved arginine on the microtubule surface. The substitution of a cysteine or histidine at the conserved arginine at position 402 (R402C and R402H, respectively) lead to lissencephaly spectrum phenotypes characterized by reduction to complete ablation of cortical folding. I explore how TUBA1A-R402C and -R402H mutations alter microtubule function using a multi-system approach that spans in utero electroporation of embryonic mouse brains to assess tissue-level defects in neuronal migration, primary rat neurons to assess cellular defects, and budding yeast to interrogate specific tubulin functions. I discovered that ectopic expression of TUBA1A-R402C and -R402H patient alleles disrupts cortical neuron positioning in the developing mouse brain. This provides the first evidence that these patient mutations are not only causal for the incorrect neuron positioning associated with lissencephaly phenotypes, but also demonstrates the dominant nature of TUBA1A-R402C/H mutations in a system where adequate levels of wild-type, endogenous Tuba1a are present. At the molecular level, I demonstrated using the budding yeast system that R402C/H mutants selectively impair dynein motors, without impairing kinesins or grossly altering microtubule stability. Further, my work has revealed that the level of dynein disruption scales with the cellular abundance of mutant protein in the cells, as a more significant dynein defect is observed in yeast cells containing a higher ratio of mutant to wild-type alleles. In addition to investigating the mechanism of the most common TUBA1A mutations to R402, I explore the impact of mutations to conserved residue V409. Depending on the substituted side-chain, V409A or V409I, patients present with brain malformations of drastically different severity. Here, I show that V409A and V409I mutations both dominantly disrupt cortical migration, but that the migration phenotype correlates with the observed patient outcome. Finally, in this dissertation I build upon my data as well as current tubulinopathy literature to propose that the primary mechanism of tubulinopathy mutants is not haploinsufficiency, but involves populating the microtubule network with dominant mutant tubulin deficient for specific microtubule functions. This hypothesis is supported by data generated using the model system budding yeast, where I interrogate how a panel of TUBA1A mutations that lead to distinct brain malformations impact tubulin incorporation and gross microtubule function. This work provides the strongest evidence to date that tubulin mutations associated with disease are causal for neuronal migration disorders that underlie brain malformation, and supports a new model where mutant tubulins generate poisoned microtubule networks in young neurons.