Giant Ankyrins Awake: Select Roles of Giant Ankyrins in Axons of the Central Nervous System

Organization of membrane domains in axons is key for the normal transmission of information from one neuron to another. Giant ankyrin proteins are two such members of axonal membrane domains, where giant ankyrin-B (440kDa) is localized to the plasma membrane of axons and giant ankyrin-G (480kDa) is localized to excitable membrane domains of axons as well as somatodendritic regions later in development. Mutations in these proteins are found in autistic individuals (giant ankyrin-B) and bipolar and schizophrenia patients (giant ankyrin-G). However, the function of giant ankyrin-B and the regulation of giant ankyrin-G remain elusive. Here we developed two transgenic mouse lines lacking giant ankyrin-B: one that completely eliminates giant ankyrin-B by Cre-dependent removal of the inserted sequence, and another CRISPR induced frameshift inside of the inserted sequence that mimics the variant in one autistic individual, to decipher its role in the central nervous system. Using analyses of cellular, brain-wide connectivity, and behavioral assays of these transgenic mice, we show the L1CAM-giant ankyrin-B complex is essential for normal axon branching and brain connectivity. Perturbations to brain connectivity by giant ankyrin-B mutation leads to selective deficits in pup ultrasonic vocalizations, male territory marking, and increased plasticity for reversal learning in a binary water T-maze. Secondly, I developed a novel solubilization technique for the denatured isolation of previously biochemically inaccessible giant ankyrin proteins. With this technique, we determined that phosphorylation was the major post-translational modification on giant ankyrin-G, and also on beta-4 spectrin sigma six isoforms. The stoichiometry of the phosphorylation was increased in these two excitable membrane proteins in comparison to the other isoforms of these proteins. By comparing developmental stages, there is a trend of decreased phosphorylation of giant ankyrin proteins within the giant inserted region during maturation of excitable membrane domains. Finally, we show a developmental change in ankyrin-G polypeptide levels and pinceau synapse formation on Purkinje cells in a mouse model with a disrupted ankyrin-G/GABARAP binding site. Together, these findings demonstrate that giant ankyrin proteins are critical proteins for the normal connectivity and conductance of neurons and that they are dynamically regulated through post-translational phosphorylation.