Authors: Courtney Johnson
In a study carried out at the RIKEN Brain Science Institute (Wako City, Saitama, Japan), researchers have utilized both transfected cell-expression systems and in vivo mouse models to investigate the effect of a novel mutation on the abnormal axonal growth of neurons characteristic of congenital fibrosis of the extraocular muscles type 3 (CFEOM3). The findings were published recently in Nature Communications.
The research team introduced a new mutation into specific kinesin proteins in order to examine its impact on the kinesin interaction with β3-tubulin – the protein containing the disease-causing mutation in CFEOM3. The abnormal axonal growth observed in CFEOM3 caused by mutated β3-tubulin was seen to be suppressed by the introduction of this new mutation in the kinesin protein as the novel mutation restored motor activity of kinesin along microtubules, thus restoring axonal growth to normal levels.
CFEOM3 is a congenital ocular motility disease with characteristic abnormal axonal growth due to debilitated axon guidance and maintenance that affects the muscles governing eye movement. This abnormal growth is associated with mutations in β3-tubulin, a protein incorporated into microtubules and involved in intracellular transport and structure in the developing nervous system. A mutation in human β3-tubulin (TUBB3) is an established cause of CFEOM3, with R262 being the most common amino acid mutation.
In CFEOM, the R262 mutation in TUBB3 is vital in mediating the kinesin–tubulin interaction and thus axonal growth and development. “Our original aim was to understand the molecular mechanism underlying kinesin–tubulin interaction in the pathogenic mutants,” explains Itsushi Minoura (RIKEN Brain Science Institute). “Surprisingly, the kinesin mutants that we created effectively rescued kinesin motility and axonal growth, even in live mice.”
Researchers utilized a baculovirus-insect cell-expression system in vitro and found that the binding of kinesins to microtubules was inhibited (particularly in the kinesin KIF5B) when the R262 CFEOM3-causing mutation in β3-tubulin is present.
By identifying the area of the kinesin protein that interacts with the β3-tubulin microtubule, it was discovered that the kinesin KIF5B binds to β3-tubulin at the amino acid D279. Researchers then introduced a mutation to replace D279 with D279R, which allowed the kinesin protein to attach and move along the R262-mutated β3-tubulin.
In following experiments R262-mutant β3-tubulin was transfected into disassociated embryonic neurons and then cotransfected with the kinesin D279R mutant in order to examine if the D279R kinesin mutant would be capable of the same suppressive activity on axonal growth in cell cultures. Results demonstrated that axonal growth in neurons cotransfected with both mutants was successfully suppressed.
An in vivo model of mouse embryos was also utilized in the study, employing in utero electroporation to transfect the postulated disease-correcting mutant of another kinesin (D325R KIF2A). Both kinesin mutants (KIF2A and KIF5B) were observed to effectively suppress abnormal growth caused by the R262 CFEOM disease-causing mutation.
Minoura commented further:”while the rescue experiment is not available for humans, understanding the roles of the many types of tubulin in normal mammalian brain development is an important step towards understanding the pathogenesis of many neurodevelopmental disorders such as lissencephaly and polymicroglia.”
Sources: RIKEN Brain Science Institute Press Release; Minoura I, Takazaki H, Ayukawa R et al. Reversal of axonal growth defects in an extraocular fibrosis model by engineering the kinesin–microtubule interface. Nat. Commun. 7, 10058 (2016).