Novel α-tubulin mutation disrupts neural development and tubulin proteostasis

Abstract

Mutations in the microtubule cytoskeleton are linked to cognitive and locomotor defects during development, and neurodegeneration in adults. How these mutations impact microtubules, and how this alters function at the level of neurons is an important area of investigation. Using a forward genetic screen in mice, we identified a missense mutation in Tuba1a α-tubulin that disrupts cortical and motor neuron development. Homozygous mutant mice exhibit cortical dysgenesis reminiscent of human tubulinopathies. Motor neurons fail to innervate target muscles in the limbs and show synapse defects at proximal targets. To directly examine effects on tubulin function, we created analogous mutations in the α-tubulin isotypes in budding yeast. These mutations sensitize yeast cells to microtubule stresses including depolymerizing drugs and low temperatures. Furthermore, we find that mutant α-tubulin is depleted from the cell lysate and from microtubules, thereby altering ratios of α-tubulin isotypes. Tubulin-binding cofactors suppress the effects of the mutation, indicating an important role for these cofactors in regulating the quality of the α-tubulin pool. Together, our results give new insights into the functions of Tuba1a, mechanisms for regulating tubulin proteostasis, and how compromising these may lead to neural defects.

Tubulin mutations in brain development disorders: Why haploinsufficiency does not explain TUBA1A tubulinopathies

“…The Tuba1a ‐S140G mutation impairs the ability of α‐tubulin to bind GTP, which disrupts tubulin heterodimer formation and reduces the functional tubulin pool (Keays et al, ). The Tuba1a ‐N102D substitution also destabilizes the heterodimer and impairs incorporation of mutant Tuba1a into the microtubule lattice (Gartz Hanson et al, ). However, the divergent neurodevelopmental and behavioral phenotypes associated with the heterozygous S140G and N102D mutant mice suggest that these variants may impact neuronal microtubule function via several differing mechanisms, not solely through loss‐of‐function.…”

“…Tuba1a-N102D homozygous mice have dramatic defects in brain development including abnormal cortical lamination and reduction in the size of olfactory bulbs, indicative of impaired neuronal migration (Gartz Hanson et al, 2016). In contrast, Tuba1a-N102D heterozygous mice are viable, undergo normal cortical lamination, and do not exhibit any evidence of neuronal migration errors (Gartz Hanson et al, 2016). From a mechanistic standpoint, Tuba1a-R215*, -S140G and -N102D variants are all predicted to reduce the levels of functional Tuba1a, despite the observed difference in heterozygous phenotypes (Bittermann et al, 2019;Gartz Hanson et al, 2016;Keays et al, 2007).…”

Modeling disease-correlated TUBA1A mutation in budding yeast reveals a molecular basis for tubulin dysfunction

“…The reduced incorporation of the mutant tubulin into microtubules appears to be compensated by a corresponding increase in Tub3 incorporation in the tub1 G437R mutant, and explains the reliance of tub1 G437R mutant cells on Tub3 expression. Previous studies estimated that Tub3 accounts for roughly ~10-30% of the cell's α-tubulin content (Bode et al, 2003;Gartz Hanson et al, 2016).…”

“…6D). It is interesting to note that one of these studies (Gartz Hanson et al, 2016) observed a ~17% increase in the rate of microtubule polymerization that was dependent on the presence of a wild-type copy of Tub3. This suggests that the increased proportion of Tub3 in the mutant microtubules may be partially responsible for the altered dynamicity observed here (16% increase in polymerization rate; Fig.…”

“…Each of these 4 has a distinct expression pattern, and thus every cell's tubulin content is a composite mixture of these many variants. In contrast to higher eukaryotes, things are much simpler in the budding yeast Saccharomyces cerevisiae in which ~70-90% of α-tubulin is expressed from the essential TUB1 gene, with the remaining ~10-30% arising from the TUB3 gene (Bode et al, 2003;Gartz Hanson et al, 2016;Schatz et al, 1986b). In addition to its simplicity, the mechanisms and effectors of spindle orientation (e.g., dynein, Lis1) and microtubule dynamics and function (e.g., tubulin, EB1, CLIP-170, ChTOG) are highly conserved between humans and budding yeast.…”