Neuronal development proceeds through several stages, such as axon and dendrite differentiation, elongation, branching, and pathfinding. Extracellular guidance cues play an essential role in these processes. Activation of downstream signaling of guidance receptors eventually leads to the cytoskeleton rearrangement. Microtubules (MTs), as one form of the cytoskeleton, play an important role in axon and dendrite outgrowth, elongation and branching. Netrin-1, a canonical guidance molecule, binds to its receptors Deleted in Colorectal Cancer (DCC), Down Syndrome Cell Adhesion Molecule (DSCAM) and uncoordinated-5 (UNC5) mediating neuronal development. Our recent studies have shown that TUBB3, a neuronal ß-tubulin isotype III, directly binds to DCC and Netrin-1 induces this interaction. Results from multiple function assays indicate that TUBB3 is specifically involved in Netrin-1-induced axon outgrowth and guidance.
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. Here, we provide evidence that TUBB3 mutations impair Netrin/DCC signaling in the developing nervous system. The interaction of DCC with most of 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 enhances the interaction of endogenous DCC with wild type human TUBB3, but not with R262C or A302V, in primary neurons. Netrin-1 also increases the co-sedimentation of DCC with polymerized MTs in primary neurons expressing 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 projection and pathfinding in ovo. Our study reveals that missense TUBB3 mutations specifically disrupt Netrin/DCC-mediated attractive signaling.
MT dynamics play an important role in Netrin-1-promoted axon outgrowth, branching, and axon pathfinding. However, the mechanism by which Netrin-1 regulates this process is not clear. The MT-associated protein (MAP) tau regulates MT stability and dynamics, which are important for neuronal development in the nervous system. Our study shows that tau interacts with the Netrin receptor DCC, and Netrin-1 induces this interaction in primary neurons. Tau colocalizes with DCC in the growth cone of primary neurons and Netrin-1 induces this colocalization. Activation of JNK, GSK-3 and Src family kinases are important for Netrin-1-induced DCC/tau interaction. Knockdown of tau inhibits Netrin-1-induced axon outgrowth, branching and commissural axon attraction in vitro and leads to defects in commissural axon projection in the chick spinal cord in vivo. These findings suggest that tau is involved in Netrin-1 signaling and essential for Netrin-1-promoted neuronal development.
In general, these studies are focusing on the role of MT component protein TUBB3 and MT-associated protein tau in the Netrin-1 signaling. The study of TUBB3 mutants further validates the essential role of TUBB3 in Netrin-1-mediated neuronal development by showing that TUBB3 mutants A302V and R262C found in patients disrupt the function of TUBB3 in Netrin-1-mediated neurite outgrowth, axon branching and attraction. The study of tau reveals that tau is involved in Netrin-1-mediated neuronal development. However, the questions regarding the relationship between TUBB3 and tau in Netrin signaling need to be further addressed. For instance, whether Netrin-1 regulates the interaction between tau and TUBB3, and whether the interaction between DCC and tau is dependent upon the presence of TUBB3 need to be clarified.