Reversal of axonal growth defects in an extraocular fibrosis model by engineering the kinesin-microtubule interface.

Abstract

Mutations in human β3-tubulin (TUBB3) cause an ocular motility disorder termed congenital fibrosis of the extraocular muscles type 3 (CFEOM3). In CFEOM3, the oculomotor nervous system develops abnormally due to impaired axon guidance and maintenance; however, the underlying mechanism linking TUBB3 mutations to axonal growth defects remains unclear. Here, we investigate microtubule (MT)-based motility in vitro using MTs formed with recombinant TUBB3. We find that the disease-associated TUBB3 mutations R262H and R262A impair the motility and ATPase activity of the kinesin motor. Engineering a mutation in the L12 loop of kinesin surprisingly restores a normal level of motility and ATPase activity on MTs carrying the R262A mutation. Moreover, in a CFEOM3 mouse model expressing the same mutation, overexpressing the suppressor mutant kinesin restores axonal growth in vivo. Collectively, these findings establish the critical role of the TUBB3-R262 residue for mediating kinesin interaction, which in turn is required for normal axonal growth and brain development.