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Axon–axon interactions are essential for axon guidance during nervous system wiring. However, it is unknown whether and how the growth cones communicate with each other while sensing and responding to guidance cues. We found that the Parkinson’s disease gene, leucine-rich repeat kinase 2 (LRRK2), has an unexpected role in growth cone–growth cone communication. The LRRK2 protein acts as a scaffold and induces Frizzled3 hyperphosphorylation indirectly by recruiting other kinases and also directly phosphorylates Frizzled3 on threonine 598 (T598). In LRRK1 or LRRK2 single knockout, LRRK1/2 double knockout, and LRRK2 G2019S knockin, the postcrossing spinal cord commissural axons are disorganized and showed anterior–posterior guidance errors after midline crossing. Growth cones from either LRRK2 knockout or G2019S knockin mice showed altered interactions, suggesting impaired communication. Intercellular interaction between Frizzled3 and Vangl2 is essential for planar cell polarity signaling. We show here that this interaction is regulated by phosphorylation of Frizzled3 at T598 and can be regulated by LRRK2 in a kinase activity-dependent way. In the LRRK1/2 double knockout or LRRK2 G2019S knockin, the dopaminergic axon bundle in the midbrain was significantly widened and appeared disorganized, showing aberrant posterior-directed growth. Our findings demonstrate that LRRK2 regulates growth cone–growth cone communication in axon guidance and that both loss-of-function mutation and a gain-of-function mutation (G2019S) cause axon guidance defects in development.

The development of neural circuits relies on axon projections establishing diverse, yet well-defined, connections between areas of the nervous system. Each projection is formed by growth cones—subcellular specializations at the tips of growing axons, encompassing sets of molecules that control projection-specific growth, guidance, and target selection 1 . To investigate the set of molecules within native growth cones that form specific connections, here we developed growth cone sorting and subcellular RNA–proteome mapping, an approach that identifies and quantifies local transcriptomes and proteomes from labelled growth cones of single projections in vivo. Using this approach on the developing callosal projection of the mouse cerebral cortex, we mapped molecular enrichments in trans-hemispheric growth cones relative to their parent cell bodies, producing paired subcellular proteomes and transcriptomes from single neuron subtypes directly from the brain. These data provide generalizable proof-of-principle for this approach, and reveal molecular specializations of the growth cone, including accumulations of the growth-regulating kinase mTOR 2 , together with mRNAs that contain mTOR-dependent motifs 3 , 4 . These findings illuminate the relationships between subcellular distributions of RNA and protein in developing projection neurons, and provide a systems-level approach for the discovery of subtype- and stage-specific molecular substrates of circuit wiring, miswiring, and the potential for regeneration. A subcellular sorting approach enables quantitative analysis of subtypes of growth cones in the brain, and reveals subcellular relationships between local mRNA and local proteomes in developing projection neurons.

The neurotransmitter serotonin has been implicated in a range of complex neurological disorders linked to alterations of neuronal circuitry. Serotonin is synthesized in the developing brain before most neuronal circuits become fully functional, suggesting that serotonin might play a distinct regulatory role in shaping circuits prior to its function as a classical neurotransmitter. In this study, we asked if serotonin acts as a guidance cue by examining how serotonin alters growth cone motility of rodent sensory neurons in vitro. Using a growth cone motility assay, we found that serotonin acted as both an attractive and repulsive guidance cue through a narrow concentration range. Extracellular gradients of 50 µM serotonin elicited attraction, mediated by the serotonin 5-HT2a receptor while 100 µM serotonin elicited repulsion mediated by the 5-HT1b receptor. Importantly, high resolution imaging of growth cones indicated that these receptors signalled through their canonical pathways of endoplasmic reticulum-mediated calcium release and cAMP depletion, respectively. This novel characterisation of growth cone motility in response to serotonin gradients provides compelling evidence that secreted serotonin acts at the molecular level as an axon guidance cue to shape neuronal circuit formation during development.

Chondroitin sulfate proteoglycans (CSPGs) represent a major barrier to regenerating axons in the central nervous system (CNS), but the structural diversity of their polysaccharides has hampered efforts to dissect the structure-activity relationships underlying their physiological activity. By taking advantage of our ability to chemically synthesize specific oligosaccharides, we demonstrate that a sugar epitope on CSPGs, chondroitin sulfate-E (CS-E), potently inhibits axon growth. Removal of the CS-E motif significantly attenuates the inhibitory activity of CSPGs on axon growth. Furthermore, CS-E functions as a protein recognition element to engage receptors including the transmembrane protein ty rosi ne phosphatase PTPσ, thereby triggering downstream pathways that inhibit axon growth. Finally, masking the CS-E motif using a CS-Especific antibody reversed the inhibitory activity of CSPGs and stimulated axon regeneration in vivo. These results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides can direct important physiological processes and provide new therapeutic strategies to regenerate axons after CNS injury.

Cells sense the environment's mechanical stiffness to control their own shape, migration, and fate. To better understand stiffness sensing, we constructed a stochastic model of the \"motor-clutch\" force transmission system, where molecular clutches link F-actin to the substrate and mechanically resist myosin-driven F-actin retrograde flow. The model predicts two distinct regimes: (i) \"frictional slippage,\" with fast retrograde flow and low traction forces on stiff substrates and (ii) oscillatory \"load-and-fail\" dynamics, with slower retrograde flow and higher traction forces on soft substrates. We experimentally confirmed these model predictions in embryonic chick forebrain neurons by measuring the nanoscale dynamics of single-growth-cone filopodia. Furthermore, we experimentally observed a model-predicted switch in F-actin dynamics around an elastic modulus of 1 kilopascal. Thus, a motor-clutch system inherently senses and responds to the mechanical stiffness of the local environment.

Herpes simplex virus type 1 (HSV-1) has evolved mechanisms to exploit the host cytoskeleton during entry, replication and exit from cells. In this study, we determined the role of actin and the molecular motor proteins, myosin II and myosin V, in the transport and release of HSV-1 from axon termini, or growth cones. Using compartmentalized neuronal devices, we showed that inhibition of actin polymerization, but not actin branching, significantly reduced the release of HSV-1 from axons. Furthermore, we showed that inhibition of myosin V, but not myosin II, also significantly reduced the release of HSV-1 from axons. Using confocal and electron microscopy, we determined that viral components are transported along axons to growth cones, despite actin or myosin inhibition. Overall, our study supports the role of actin in virus release from axonal growth cones and suggests myosin V as a likely candidate involved in this process.

The growth cone guides the axon or dendrite of striatal GABAergic projection neurons that protrude into the midbrain and cortex and form complex neuronal circuits and synaptic networks in a developing brain, aberrant projections and synaptic connections in the striatum related to multiple brain disorders. Previously, we showed that ketamine, an anesthetic, reduced dendritic growth, dendritic branches, and spine density in human striatal GABAergic neurons. However, whether ketamine affects the growth cone, the synaptic connection of growing striatal GABAergic neurons has not been tested. Using human GABAergic projection neurons derived from human inducible pluripotent stem cells (hiPSCs) and embryonic stem cells (ES) in vitro, we tested ketamine effects on the growth cones and synapses in developing GABAergic neurons by assessing the morphometry and the glycogen synthase kinase-3 (GSK-3) and histone deacetylase 6 (HDAC6) pathway. Ketamine exposure impairs growth cone formation, synaptogenesis, dendritic development, and maturation via ketamine-mediated activation of GSK-3 pathways and inhibiting HDAC6, an essential stabilizing protein for dendritic morphogenesis and synapse maturation. Our findings identified a novel ketamine neurotoxic pathway that depends on GSK-3β and HDAC6 signaling, suggesting that microtubule acetylation is a potential target for reducing ketamine’s toxic effect on GABAergic projection neuronal development.

Recent studies in vertebrates and Caenorhabditis elegans have reshaped models of how the axon guidance cue UNC-6/Netrin functions in dorsal-ventral axon guidance, which was traditionally thought to form a ventral-to-dorsal concentration gradient that was actively sensed by growing axons. In the vertebrate spinal cord, floorplate Netrin1 was shown to be largely dispensable for ventral commissural growth. Rather, short range interactions with Netrin1 on the ventricular zone radial glial stem cells was shown to guide ventral commissural axon growth. In C. elegans , analysis of dorsally-migrating growth cones during outgrowth has shown that growth cone polarity of filopodial extension is separable from the extent of growth cone protrusion. Growth cones are first polarized by UNC-6/Netrin, and subsequent regulation of protrusion by UNC-6/Netrin is based on this earlier-established polarity (the Polarity/Protrusion model). In both cases, short-range or even haptotactic mechanisms are invoked: in vertebrate spinal cord, interactions of growth cones with radial glia expressing Netrin-1; and in C. elegans, a potential close-range interaction that polarizes the growth cone. To explore potential short-range and long-range functions of UNC-6/Netrin, a potentially membrane-anchored transmembrane UNC-6 (UNC-6(TM)) was generated by genome editing. unc-6(tm) was hypomorphic for dorsal VD/DD axon pathfinding, indicating that it retained some unc-6 function. Polarity of VD growth cone filopodial protrusion was initially established in unc-6(tm) , but was lost as the growth cones migrated away from the unc-6(tm) source in the ventral nerve cord. In contrast, ventral guidance of the AVM and PVM axons was equally severe in unc-6(tm) and unc-6(null) . Together, these results suggest that unc-6(tm) retains short-range functions but lacks long-range functions due to reduced secreted UNC-6. Ectopic unc-6(+) expression from non-ventral sources did not dramatically perturb dorsal VD growth cone polarity or axon outgrowth, suggesting that ectopic UNC-6 cannot redirect polarity once it is established in the VD/DD neurons. This is not what would be expected of a growth cone dynamically reading a gradient of UNC-6, but is consistent with the Polarity/protrusion model of growth cone guidance away from UNC-6/Netrin.

Micro and nanoscale patterning of surface features and biochemical cues have emerged as tools to precisely direct neurite growth into close proximity with next generation neural prosthesis electrodes. Biophysical cues can exert greater influence on neurite pathfinding compared to the more well studied biochemical cues; yet the signaling events underlying the ability of growth cones to respond to these microfeatures remain obscure. Intracellular Ca 2+ signaling plays a critical role in how a growth cone senses and grows in response to various cues (biophysical features, repulsive peptides, chemo-attractive gradients). Here, we investigate the role of inositol triphosphate (IP3) and ryanodine-sensitive receptor (RyR) signaling as sensory neurons (spiral ganglion neurons, SGNs, and dorsal root ganglion neurons, DRGNs) pathfind in response to micropatterned substrates of varied geometries. We find that IP3 and RyR signaling act in the growth cone as they navigate biophysical cues and enable proper guidance to biophysical, chemo-permissive, and chemo-repulsive micropatterns. In response to complex micropatterned geometries, RyR signaling appears to halt growth in response to both topographical features and chemo-repulsive cues. IP3 signaling appears to play a more complex role, as growth cones appear to sense the microfeatures in the presence of xestospongin C but are unable to coordinate turning in response to them. Overall, key Ca 2+ signaling elements, IP3 and RyR, are found to be essential for SGNs to pathfind in response to engineered biophysical and biochemical cues. These findings inform efforts to precisely guide neurite regeneration for improved neural prosthesis function, including cochlear implants.

Filopodial dynamics are thought to control growth cone guidance, but the types and roles of growth cone dynamics underlying neural circuit assembly in a living brain are largely unknown. To address this issue, we have developed long-term, continuous, fast and high-resolution imaging of growth cone dynamics from axon growth to synapse formation in cultured Drosophila brains. Using R7 photoreceptor neurons as a model we show that >90% of the growth cone filopodia exhibit fast, stochastic dynamics that persist despite ongoing stepwise layer formation. Correspondingly, R7 growth cones stabilize early and change their final position by passive dislocation. N-Cadherin controls both fast filopodial dynamics and growth cone stabilization. Surprisingly, loss of N-Cadherin causes no primary targeting defects, but destabilizes R7 growth cones to jump between correct and incorrect layers. Hence, growth cone dynamics can influence wiring specificity without a direct role in target recognition and implement simple rules during circuit assembly. Genes encode complicated developmental processes, but it is clear that genetic information cannot encode each and every individual connection that forms between the nerve cells in a brain. Instead, the individual cells and nerve endings must make decisions during brain development. Up until now, few examples were known for how these nerve endings move and choose their paths and partners in a living, developing brain. The fruit fly Drosophila provides a useful model to explore the ‘wiring’ of nerve cells in the brain, partly because a fruit fly’s brain develops within a few days. However, most previous studies have relied on identifying mutant flies with disrupted brain wiring and studying them using still images. Now, Özel et al. have developed a new imaging method that has enough resolution and speed over sufficiently long periods to track the growing nerve endings in a developing fly brain. The method was applied to a model nerve cell in the fly’s visual system. This revealed that most of this nerve’s dynamic changes are short-lived and random, and appear to help to stabilize the developing nerve ending, rather than guide it to a target. Özel et al. also found that a protein called N-Cadherin, previously thought to be required for the targeting of developing nerve endings, actually plays a role in their stabilization. These findings uncover the roles of changes in nerve endings during long-term brain development; this was previously largely unknown for any organism. The next stage in this research will involve further analyses of both wild type and mutant flies to try and work out general principles about how the brain develops via the decoding of genetic information.