부산대학교 도서관

Microtubules play a critical role in multiple aspects of neurodevelopment, including the generation, migration and differentiation of neurons. A recurrent mutation (R402H) in the α-tubulin gene TUBA1A is known to cause lissencephaly with cerebellar and striatal phenotypes. Previous work has shown that this mutation does not perturb the chaperone-mediated folding of tubulin heterodimers, which are able to assemble and incorporate into the microtubule lattice. To explore the molecular mechanisms that cause the disease state we generated a new conditional mouse line that recapitulates the R402H variant. We show that heterozygous mutants present with laminar phenotypes in the cortex and hippocampus, as well as a reduction in striatal size and cerebellar abnormalities. We demonstrate that homozygous expression of the R402H allele causes neuronal death and exacerbates a cell intrinsic defect in cortical neuronal migration. Microtubule sedimentation assays coupled with quantitative mass spectrometry demonstrated that the binding and/or levels of multiple microtubule associated proteins (MAPs) are perturbed by the R402H mutation including VAPB, REEP1, EZRIN, PRNP and DYNC1l1/2. Consistent with these data we show that the R402H mutation impairs dynein-mediated transport which is associated with a decoupling of the nucleus to the microtubule organising center. Our data support a model whereby the R402H variant is able to fold and incorporate into microtubules, but acts as a gain of function by perturbing the binding of MAPs. Author summary: Microtubules are polymers composed of tubulin proteins, which play an important role in the development of the human brain. Genetic mutations in tubulin genes are known to cause neurodevelopmental diseases, including lissencephaly which is characterised by an impairment in the migration of neurons. In this study we investigate how a common mutation (R402H) in TUBA1A causes lissencephaly by generating and characterising a mouse with the same variant. We show that affected animals recapitulate multiple aspects of the human disease; including laminar perturbations in the cortex and hippocampus, attributable to defects in neuronal migration at key developmental time points. To characterize the molecular implications of the R402H mutation we purified microtubules from the developing brain, and analysed the proteins present by mass spectrometry. This revealed that the binding of DYNC1I1/2 to microtubules is altered in mice with the R402H mutation. Our results provide insight into the molecular pathology underlying tubulin related disease states, and provide a foundation for the rational design of therapeutic interventions. [ABSTRACT FROM AUTHOR]