Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

Area of Science:

• Neuroscience
• Molecular Biology
• Developmental Biology

Background:

• Heterozygous missense mutations in the human TUBB3 gene are linked to brain malformations, including defects in axon guidance, neuronal migration, and differentiation.
• The precise molecular mechanisms underlying these mutation-related axon guidance abnormalities remain largely unknown.
• Netrin-1, a key guidance cue, interacts with TUBB3 and its receptor deleted in colorectal cancer (DCC), and TUBB3 is essential for netrin-1-mediated axon development.

Purpose of the Study:

• To investigate how TUBB3 mutations affect netrin/DCC signaling pathways in the developing nervous system.
• To elucidate the molecular basis of impaired axon guidance caused by TUBB3 mutations.

Main Methods:

• Assessed the interaction between wild-type TUBB3 and its mutants with DCC in primary neurons.
• Utilized co-localization studies to examine the subcellular distribution of TUBB3 mutants and DCC in neuronal growth cones.
• Investigated the effect of netrin-1 on the interaction of TUBB3 with DCC and the co-sedimentation of DCC with polymerized microtubules (MTs).
• Evaluated the functional impact of TUBB3 mutations on netrin-1-induced neurite outgrowth, branching, and attraction in vitro.
• Examined the in ovo effects of TUBB3 mutations on spinal cord commissural axon (CA) projection and pathfinding.

Main Results:

• Most TUBB3 mutants (8/12) showed significantly reduced interaction with DCC compared to wild-type TUBB3.
• Specific mutants (R262C, A302V) displayed decreased co-localization with DCC in growth cones and failed to enhance DCC-MT interaction upon netrin-1 stimulation.
• Expression of R262C or A302V mutants suppressed netrin-1-induced neurite outgrowth, branching, and attraction in vitro.
• These mutants also induced defects in spinal cord commissural axon (CA) projection and pathfinding in ovo.

Conclusions:

• Missense TUBB3 mutations specifically disrupt the netrin/DCC-mediated attractive signaling pathway.
• These disruptions in signaling contribute to the observed brain malformations and axon guidance defects associated with TUBB3 mutations.
• The findings provide crucial insights into the molecular mechanisms underlying neurodevelopmental disorders linked to TUBB3.