Deciphering molecular pathomechanisms of alpha-tubulin mutation-based neuronal disorders.

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Nerve cells electrically wire the nervous system throughout the body via cable-like protrusions called axons. They have to be maintained for a lifetime. Therefore, damage to axons through accidents or their loss in neurodegenerative diseases has a devastating impact on neuronal function.The actin and microtubule (MT) cytoskeleton forms the structural backbone of axons; MTs in particular, provide life-sustaining cellular transport highways. Known mutations in human alpha-tubulins lead to lissencephaly; where axonal outgrowth, neuronal migration and guidance are affected. However, the molecular pathomechanisms of these mutations are poorly understood. The proposed project aims to clarify these pathomechanisms as well as changes in protein networks downstream of alpha1-Tubulin-mutations, using Drosophila neuronal tissue and in vivo analysis. alpha1-Tubulin is strongly conserved (97% identity between human and fly versions); therefore, identified protein interactions are expected to be highly translational from flies to humans. Specific lissencephaly-related alpha1-tubulin amino acid site mutations will be generated in flies and used to study their impact at a molecular, cellular and organismic level. To that end, a genetic null background of the Drosophila neuronal alpha1-tubulin gene will be generated. Using CRISPR-Cas9-homology directed repair, a PhiC31/attP landing platform will replace the endogenous gene locus. This will subsequently allow the easy reintroduction of alpha1-tubulins with varying engineered amino acid substitution mutations via recombinase mediated cassette exchange. At the cellular level, immunohistochemical and biochemical stainings, real-time qRT-PCR, live cell imaging and genetic interactions studies will be used to determine microtubule function in primary neuron cultures expressing the mutated alpha1-tubulin versions. To that end, microtubule stability, dynamics, posttranslational modifications, axonal transport and microtubule binding protein interactions with MTs. will be analysed. At an organismic level, axon guidance, fasciculation, neuronal growth and neuronal function will be studied during animal development and aging. This will be achieved by using the UAS-Gal4 and clonal MARCM systems for the expression of the mutated alpha1-tubulin versions in combination with advanced immunohistochemical imaging techniques and behavioural assays.The proposed research strategy will unravel changes in protein interactions with microtubules in alpha1-tubulin mutation based neuronal disorders. This research strategy can be easily adapted in the future to study the importance of additional posttranslational modifications found on tubulins, to analyse molecular aspects of lissencephaly-linked mutations in non-neuronal tissues, and to identify new molecular targets in tubulin mutation-linked disorders and neurodegenerative diseases.