Heterozygous missense mutations in human TUBB3 gene result in a spectrum of brain malformations associated with defects in axon guidance, neuronal migration and differentiation. However, the molecular mechanisms underlying mutation-related axon guidance abnormalities are unclear. Recent studies have shown that netrin-1, a canonical guidance cue, induced the interaction of TUBB3 with the netrin receptor deleted in colorectal cancer (DCC). Furthermore, TUBB3 is required for netrin-1-induced axon outgrowth, branching and pathfinding. Here, we provide evidence that TUBB3 mutations impair netrin/DCC signaling in the developing nervous system. The interaction of DCC with most TUBB3 mutants (eight out of twelve) is significantly reduced compared to the wild-type TUBB3. TUBB3 mutants R262C and A302V exhibit decreased subcellular colocalization with DCC in the growth cones of primary neurons. Netrin-1 increases the interaction of endogenous DCC with wild-type human TUBB3, but not R262C or A302V, in primary neurons. Netrin-1 also increases co-sedimentation of DCC with polymerized microtubules (MTs) in primary neurons expressing the wild-type TUBB3, but not R262C or A302V. Expression of either R262C or A302V not only suppresses netrin-1-induced neurite outgrowth, branching and attraction in vitro, but also causes defects in spinal cord commissural axon (CA) projection and pathfinding in ovo. Our study reveals that missense TUBB3 mutations specifically disrupt netrin/DCC-mediated attractive signaling.

Introduction 3. Materials and methods 3.1. Test materials 3.2. Experimental animals 3.3. Study design 3.4. Suspension 3.5. Sub-acute oral gavage of the alkenones 3.6. Blood analysis 3.7. Histology 3.8. Assessment of the dermal toxicity after the sub-acute administration of alkenones 3.9. Assessment of the toxicity after the sub-chronic dermal administration of alkenones 3.10. Determination of skin water loss 3.11. Statistical analysis 4. Results 4.1. The effects of the sub-acute oral administration from alkenones 4.2. The effects of the sub-acute dermal exposure from alkenones 4.3. The effects of repeated dermal exposure from alkenones 5. Discussion 6. Acknowledgments 7. References