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.

Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion.

Modulation of microtubule (MT) dynamics is a key event of cytoskeleton remodeling in the growth cone (GC) during axon outgrowth and pathfinding. Our previous studies have shown that the direct interaction of netrin receptor DCC and DSCAM with polymerized TUBB3, a neuron-specific MT subunit in the brain, is required for netrin-1-mediated axon outgrowth, branching, and attraction. Here, we show that uncoupling of polymerized TUBB3 with netrin-1-repulsive receptor UNC5C is involved in netrin-1-mediated axonal repulsion. TUBB3 directly interacted with UNC5C and partially colocalized with UNC5C in the peripheral area of the GC of primary neurons from the cerebellar external granule layer of P2 mouse pups of both sexes. Netrin-1 reduced this interaction as well as the colocalization of UNC5C and TUBB3 in the GC. Results from the in vitro cosedimentation assay indicated that UNC5C interacted with polymerized TUBB3 in MTs and netrin-1 decreased this interaction. Knockdown of either TUBB3 or UNC5C blocked netrin-1-promoted axon repulsion in vitro and caused defects in axon projection of DRG toward the spinal cord in vivo Furthermore, live-cell imaging of end-binding protein 3 tagged with EGFP (EB3-GFP) in primary external granule layer cells showed that netrin-1 differentially increased MT dynamics in the GC with more MT growth in the distal than the proximal region of the GC during repulsion, and knockdown of either UNC5C or TUBB3 abolished the netrin-1 effect. Together, these data indicate that the disengagement of UNC5C with polymerized TUBB3 plays an essential role in netrin-1/UNC5C-mediated axon repulsion.SIGNIFICANCE STATEMENT Proper regulation of microtubule (MT) dynamics in the growth cone plays an important role in axon guidance. However, whether guidance cues modulate MT dynamics directly or indirectly is unclear. Here, we report that dissociation of UNC5C and polymerized TUBB3, the highly dynamic ß-tubulin isoform in neurons, is essential for netrin-1/UNC5C-promoted axon repulsion. These results not only provide a working model of direct modulation of MTs by guidance cues in growth cone navigation but also help us to understand molecular mechanisms underlying developmental brain disorders associated with TUBB3 mutations.

Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance.

The coupling of axon guidance cues, such as netrin-1, to microtubule (MT) dynamics is essential for growth cone navigation in the developing nervous system. However, whether axon guidance signaling regulates MT dynamics directly or indirectly is unclear. Here, we report that TUBB3, the most dynamic ß-tubulin isoform in neurons, directly interacts with the netrin receptor DCC, and that netrin-1 induces this interaction in primary neurons. TUBB3 colocalizes with DCC in the growth cones of primary neurons and MT dynamics is required for netrin-1-promoted association of TUBB3 with DCC. Netrin-1 not only increases co-sedimentation of DCC with polymerized MT, but also promotes MT dynamics in the growth cone. Knocking down TUBB3 inhibits netrin-1-induced MT dynamics, axon outgrowth and attraction in vitro and causes defects in commissural axon projection in the embryo. These results indicate that TUBB3 directly links netrin signaling pathways to MT dynamics and plays an important role in guiding commissural axons in vivo.

c-Jun N-terminal kinase 1 (JNK1) is required for coordination of netrin signaling in axon guidance.

The JNK family of MAPKs is involved in a large variety of physiological and pathological processes in brain development, such as neural survival, migration, and polarity as well as axon regeneration. However, whether JNK activation is involved in axon guidance remains unknown. Here, we provide evidence indicating the JNK pathway is required for Netrin signaling in the developing nervous system. Netrin-1 increased JNK1, not JNK2 or JNK3, activity in the presence of deleted in colorectal cancer (DCC) or Down syndrome cell adhesion molecule (DSCAM), and expression of both of them further enhanced Netrin-1-induced JNK1 activity in vitro. Inhibition of JNK signaling either by a JNK inhibitor, SP600125, or expression of a dominant negative form of MKK4, a JNK upstream activator, blocked Netrin-1-induced JNK1 activation in HEK293 cells. Netrin-1 increased endogenous JNK activity in primary neurons. Netrin-1-induced JNK activation was inhibited either by the JNK inhibitor or an anti-DCC function-blocking antibody. Combination of the anti-DCC function-blocking antibody with expression of DSCAM shRNA in primary neurons totally abolished Netrin-1-induced JNK activation, whereas knockdown of DSCAM partially inhibited the Netrin-1 effect. In the developing spinal cord, phospho-JNK was strongly expressed in commissural axons before and as they crossed the floor plate, and Netrin-1 stimulation dramatically increased the level of endogenous phospho-JNK in commissural axon growth cones. Inhibition of JNK signaling either by JNK1 RNA interference (RNAi) or the JNK inhibitor suppressed Netrin-1-induced neurite outgrowth and axon attraction. Knockdown of JNK1 in ovo caused defects in spinal cord commissural axon projection and pathfinding. Our study reveals that JNK1 is important in the coordination of DCC and DSCAM in Netrin-mediated attractive signaling.

Down syndrome cell adhesion molecule (DSCAM) associates with uncoordinated-5C (UNC5C) in netrin-1-mediated growth cone collapse.

In the developing nervous system, neuronal growth cones explore the extracellular environment for guidance cues, which can guide them along specific trajectories toward their targets. Netrin-1, a bifunctional guidance cue, binds to deleted in colorectal cancer (DCC) and DSCAM mediating axon attraction, and UNC5 mediating axon repulsion. Here, we show that DSCAM interacts with UNC5C and this interaction is stimulated by netrin-1 in primary cortical neurons and postnatal cerebellar granule cells. DSCAM partially co-localized with UNC5C in primary neurons and brain tissues. Netrin-1 induces axon growth cone collapse of mouse cerebellum external granule layer (EGL) cells, and the knockdown of DSCAM or UNC5C by specific shRNAs or blocking their signaling by overexpressing dominant negative mutants suppresses netrin-1-induced growth cone collapse. Similarly, the simultaneous knockdown of DSCAM and UNC5C also blocks netrin-1-induced growth cone collapse in EGL cells. Netrin-1 increases tyrosine phosphorylation of endogenous DSCAM, UNC5C, FAK, Fyn, and PAK1, and promotes complex formation of DSCAM with these signaling molecules in primary postnatal cerebellar neurons. Inhibition of Src family kinases efficiently reduces the interaction of DSCAM with UNC5C, FAK, Fyn, and PAK1 and tyrosine phosphorylation of these proteins as well as growth cone collapse of mouse EGL cells induced by netrin-1. The knockdown of DSCAM inhibits netrin-induced tyrosine phosphorylation of UNC5C and Fyn as well as the interaction of UNC5C with Fyn. The double knockdown of both receptors abolishes the induction of Fyn tyrosine phosphorylation by netrin-1. Our study reveals the first evidence that DSCAM coordinates with UNC5C in netrin-1 repulsion.