The role of microtubule-associated protein tau in netrin-1 attractive signaling.

Direct binding of netrin receptors with dynamic microtubules (MTs) in the neuronal growth cone plays an important role in netrin-mediated axon guidance. However, how netrin-1 (NTN1) regulates MT dynamics in axon turning remains a major unanswered question. Here, we show that the coupling of netrin-1 receptor DCC with tau (MAPT)-regulated MTs is involved in netrin-1-promoted axon attraction. Tau directly interacts with DCC and partially overlaps with DCC in the growth cone of primary neurons. Netrin-1 induces this interaction and the colocalization of DCC and tau in the growth cone. The netrin-1-induced interaction of tau with DCC relies on MT dynamics and TUBB3, a highly dynamic ß-tubulin isotype in developing neurons. Netrin-1 increased cosedimentation of DCC with tau and TUBB3 in MTs, and knockdown of either tau or TUBB3 mutually blocked this effect. Downregulation of endogenous tau levels by tau shRNAs inhibited netrin-1-induced axon outgrowth, branching and commissural axon attraction in vitro, and led to defects in spinal commissural axon projection in vivo. These findings suggest that tau is a key MT-associated protein coupling DCC with MT dynamics in netrin-1-promoted axon attraction.

Disease-associated mutations in human TUBB3 disturb netrin repulsive signaling.

Missense mutations in the human TUBB3 gene cause a variety of neurological disorders associated with defects in axon guidance and neuronal migration, but the underlying molecular mechanisms are not well understood. Recent studies have shown that direct coupling of dynamic TUBB3 in microtubules with netrin receptors is required for netrin-1-mediated axon guidance, and the interaction of netrin-1 repulsive receptor UNC5C with TUBB3 is involved in netrin-1 mediated axonal repulsion. Here, we report that TUBB3 mutations perturb netrin-1/UNC5C repulsive signaling in the developing nervous system. Among twelve mutants reported in previous studies, five of them show significantly reduced interaction with UNC5C in comparison to the wild-type TUBB3. TUBB3 mutants R262C and R62Q exhibit decreased subcellular colocalization with UNC5C in the peripheral area of the growth cone of primary mouse neurons. Netrin-1 reduces the colocalization of UNC5C with wild-type TUBB3, but not TUBB3 mutants R262C or R62Q, in the growth cone. Results from the in vitro cosedimentation assay indicate that netrin-1 inhibits cosedimentation of UNC5C with polymerized microtubules in primary mouse neurons expressing the wild-type TUBB3, but not R262C or R62Q. Expression of either R262C or R62Q not only blocks netrin-1-induced growth cone collapse and axonal repulsion of primary EGL cells in vitro, but also results in axon projections defects of chicken dorsal root ganglion neurons in ovo. Our study reveals that human TUBB3 mutations specifically perturb netrin-1/UNC5C-mediated repulsion.

miR-92 Suppresses Robo1 Translation to Modulate Slit Sensitivity in Commissural Axon Guidance.

Temporospatial regulation of guidance signaling is essential for axon outgrowth and pathfinding in the developing nervous system. Regulation of Robo1 levels in commissural neurons modulates Slit sensitivity facilitating proper axon guidance. The mechanisms underlying this regulation in the vertebrate nervous system are not well understood. Here, we report that miR-92, a highly conserved microRNA (miRNA), regulates chicken Robo1 expression in commissural neurons by binding to the 3' untranslated region (3' UTR) of Robo1 mRNA. miR-92 and Robo1 are differentially expressed in the developing spinal cord. miR-92 interacts with the Robo1 3'UTR to cause translational repression, but not mRNA degradation. Disruption of the miR-92/Robo1 3' UTR interaction induces premature responsiveness to Slit2 repulsion of precrossing commissural axons (CAs) in vitro and causes CA projection defects in vivo. These results indicate that miR-92 represses Robo1 expression thereby regulating Slit sensitivity to control CA projection and midline crossing.

Human TUBB3 Mutations Disrupt Netrin Attractive Signaling.

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.