Browsing by Subject "axon initial segment"

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.

Fibroblast growth factor homologous factors (FHFs) are noncanonical members of the fibroblast growth factor family (FGFs, FGF11-FGF14) that bind directly to voltage gated sodium channels (VGSCs), thereby regulating channel activity and consequently neuronal excitability. Mutations in FGF14 cause spinocerebellar ataxia while FGF13 is a candidate for X-linked mental retardation. Since FGF13 and FGF14 are nearly identical within their respective VGSC-interacting domains, those distinct pathological consequences have generally been attributed to regional differences in expression. I have shown that FGF13 and FGF14 have non-overlapping subcellular distributions and biological roles even in hippocampal neurons where both are prominent. While both FHFs are abundant in the axon initial segment (AIS), only FGF13 is observed within the soma and dendrites. shRNA knockdown and rescue strategies showed that FGF14 regulates axonal VGSCs, while FGF13 only affects VGSCs in the somatodendritic compartment. Thus, FGF13 and FGF14 have nonredundant roles in hippocampal neurons, with FGF14 acting as an AIS-dominant positive regulator and FGF13 serving as a somatodendritic negative regulator. Both of these FHFs also perform important non-VGSC regulatory roles. FGF14 is a novel potassium channel regulator, which binds to KCNQ2 and regulates both localization and function. FGF14 is also capable of serving as a “bridge” between VGSCs and KCNQ2 thus implicating it as a broad organizer of the AIS. FGF13, on the other hand is involved in a new form of neuronal plasticity called axon initial segment structural plasticity. Knockdown of FGF13 impairs AIS structural plasticity and reduces L-type CaV current through channels known to be important to this new form of plasticity. Both of these novel non-VGSC roles are specific to the FHF in question because FGF13 does not regulate KCNQ2 whereas FGF14 knockdown does not affect AIS position. These data imply wider roles for FHFs in neuronal regulation that may contribute to differing roles in neural disease.