Molecular regulation of axon termination in mechanosensory neurons.

Spatially and temporally accurate termination of axon outgrowth, a process called axon termination, is required for efficient, precise nervous system construction and wiring. The mechanosensory neurons that sense low-threshold mechanical stimulation or gentle touch have proven exceptionally valuable for studying axon termination over the past 40 years. In this Review, we discuss progress made in deciphering the molecular and genetic mechanisms that govern axon termination in touch receptor neurons. Findings across model organisms, including Caenorhabditis elegans, Drosophila, zebrafish and mice, have revealed that complex signaling is required for termination with conserved principles and players beginning to surface. A key emerging theme is that axon termination is mediated by complex signaling networks that include ubiquitin ligase signaling hubs, kinase cascades, transcription factors, guidance/adhesion receptors and growth factors. Here, we begin a discussion about how these signaling networks could represent termination codes that trigger cessation of axon outgrowth in different species and types of mechanosensory neurons.

"Floppy brain" in congenital hydrocephalus.

Hydrocephalus is classically considered to be a disorder of altered cerebrospinal fluid (CSF) circulation, leading to the dilation of cerebral ventricles. Here, we report a clinical case of a patient who presented with fetal-onset hydrocephalus with diffusely reduced cortical and white matter volumes resulting from a genetic mutation in L1CAM, a well-known hydrocephalus disease gene involved in neuronal cell adhesion and axon development. After CSF was drained from the ventricle intraoperatively, the patient's cortical mantle collapsed and exhibited a "floppy" appearance on neuroimaging, suggesting an inability of the hydrocephalic brain to maintain its structural integrity. The case provides clinical support for altered brain biomechanical properties in human hydrocephalus and adds to the emerging hypothesis that altered brain development with secondary impact on brain structural stability may contribute to ventricular enlargement in some subsets of hydrocephalus.

Temporal-specific roles of fragile X mental retardation protein in the development of the hindbrain auditory circuit.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1+ neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1+ neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first in vivo identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.

mtIF3 is locally translated in axons and regulates mitochondrial translation for axonal growth.

BACKGROUND: The establishment and maintenance of functional neural connections relies on appropriate distribution and localization of mitochondria in neurites, as these organelles provide essential energy and metabolites. In particular, mitochondria are transported to axons and support local energy production to maintain energy-demanding neuronal processes including axon branching, growth, and regeneration. Additionally, local protein synthesis is required for structural and functional changes in axons, with nuclear-encoded mitochondrial mRNAs having been found localized in axons. However, it remains unclear whether these mRNAs are locally translated and whether the potential translated mitochondrial proteins are involved in the regulation of mitochondrial functions in axons. Here, we aim to further understand the purpose of such compartmentalization by focusing on the role of mitochondrial initiation factor 3 (mtIF3), whose nuclear-encoded transcripts have been shown to be present in axonal growth cones. RESULTS: We demonstrate that brain-derived neurotrophic factor (BDNF) induces local translation of mtIF3 mRNA in axonal growth cones. Subsequently, mtIF3 protein is translocated into axonal mitochondria and promotes mitochondrial translation as assessed by our newly developed bimolecular fluorescence complementation sensor for the assembly of mitochondrial ribosomes. We further show that BDNF-induced axonal growth requires mtIF3-dependent mitochondrial translation in distal axons. CONCLUSION: We describe a previously unknown function of mitochondrial initiation factor 3 (mtIF3) in axonal protein synthesis and development. These findings provide insight into the way neurons adaptively control mitochondrial physiology and axonal development via local mtIF3 translation.

A temporal and spatial map of axons in developing mouse prostate.

Prostate autonomic and sensory axons control glandular growth, fluid secretion, and smooth muscle contraction and are remodeled during cancer and inflammation. Morphogenetic signaling pathways reawakened during disease progression may drive this axon remodeling. These pathways are linked to proliferative activities in prostate cancer and benign prostate hyperplasia. However, little is known about which developmental signaling pathways guide axon investment into prostate. The first step in defining these pathways is pinpointing when axon subtypes first appear in prostate. We accomplished this by immunohistochemically mapping three axon subtypes (noradrenergic, cholinergic, and peptidergic) during fetal, neonatal, and adult stages of mouse prostate development. We devised a method for peri-prostatic axon density quantification and tested whether innervation is uniform across the proximo-distal axis of dorsal and ventral adult mouse prostate. Many axons directly interact with or innervate neuroendocrine cells in other organs, so we examined whether sensory or autonomic axons innervate neuroendocrine cells in prostate. We first detected noradrenergic, cholinergic, and peptidergic axons in prostate at embryonic day (E) 14.5. Noradrenergic and cholinergic axon densities are uniform across the proximal-distal axis of adult mouse prostate while peptidergic axons are denser in the periurethral and proximal regions. Peptidergic and cholinergic axons are closely associated with prostate neuroendocrine cells whereas noradrenergic axons are not. These results provide a foundation for understanding mouse prostatic axon development and organization and, provide strategies for quantifying axons during progression of prostate disease.

p39 Is Responsible for Increasing Cdk5 Activity during Postnatal Neuron Differentiation and Governs Neuronal Network Formation and Epileptic Responses.

Two distinct protein cofactors, p35 and p39, independently activate Cyclin-dependent kinase 5 (Cdk5), which plays diverse roles in normal brain function and the pathogenesis of many neurological diseases. The initial discovery that loss of p35 impairs neuronal migration in the embryonic brain prompted intensive research exploring the function of p35-dependent Cdk5 activity. In contrast, p39 expression is restricted to the postnatal brain and its function remains poorly understood. Despite the robustly increased Cdk5 activity during neuronal differentiation, which activator is responsible for enhancing Cdk5 activation and how the two distinct activators direct Cdk5 signaling to govern neuronal network formation and function still remains elusive. Here we report that p39, but not p35, is selectively upregulated by histone acetylation-mediated transcription, which underlies the robust increase of Cdk5 activity during rat and mouse neuronal differentiation. The loss of p39 attenuates overall Cdk5 activity in neurons and preferentially affects phosphorylation of specific Cdk5 targets, leading to aberrant axonal growth and impaired dendritic spine and synapse formation. In adult mouse brains, p39 deficiency results in dysregulation of p35 and Cdk5 targets in synapses. Moreover, in contrast to the proepileptic phenotype caused by the lack of p35, p39 loss leads to deficits in maintaining seizure activity and induction of immediate early genes that control hippocampal excitability. Together, our studies demonstrate essential roles of p39 in neuronal network development and function. Furthermore, our data support a model in which Cdk5 activators play nonoverlapping and even opposing roles to govern balanced Cdk5 signaling in the postnatal brain. SIGNIFICANCE STATEMENT: Neuronal network development requires tightly regulated activation of Cyclin-dependent kinase 5 (Cdk5) by two distinct cofactors, p35 and p39. Despite the well-known p35-dependent Cdk5 function, why postnatal neurons express abundant p39 in addition to p35 remained unknown for decades. In this study, we discovered that selective upregulation of p39 is the underlying mechanism that accommodates the increased functional requirement of Cdk5 activation during neuronal differentiation. In addition, we demonstrated that p39 selectively directs Cdk5 to phosphorylate protein substrates essential for axonal development, dendritic spine formation, and synaptogenesis. Moreover, our studies suggest opposing roles of p39 and p35 in synaptic Cdk5 function and epileptic responses, arguing that cooperation between Cdk5 activators maintains balanced Cdk5 signing, which is crucial for postnatal brain function.

A Feed-Forward Mechanism Involving the NOX Complex and RyR-Mediated Ca2+ Release During Axonal Specification.

Physiological levels of ROS support neurite outgrowth and axonal specification, but the mechanisms by which ROS are able to shape neurons remain unknown. Ca2+, a broad intracellular second messenger, promotes both Rac1 activation and neurite extension. Ca2+ release from the endoplasmic reticulum, mediated by both the IP3R1 and ryanodine receptor (RyR) channels, requires physiological ROS levels that are mainly sustained by the NADPH oxidase (NOX) complex. In this work, we explore the contribution of the link between NOX and RyR-mediated Ca2+ release toward axonal specification of rat hippocampal neurons. Using genetic approaches, we find that NOX activation promotes both axonal development and Rac1 activation through a RyR-mediated mechanism, which in turn activates NOX through Rac1, one of the NOX subunits. Collectively, these data suggest a feedforward mechanism that integrates both NOX activity and RyR-mediated Ca2+ release to support cellular mechanisms involved in axon development. SIGNIFICANCE STATEMENT: High levels of ROS are frequently associated with oxidative stress and disease. In contrast, physiological levels of ROS, mainly sustained by the NADPH oxidase (NOX) complex, promote neuronal development and axonal growth. However, the mechanisms by which ROS shape neurons have not been described. Our work suggests that NOX-derived ROS promote axonal growth by regulating Rac1 activity, a molecular determinant of axonal growth, through a ryanodine receptor (RyR)-mediated Ca2+ release mechanism. In addition, Rac1, one of the NOX subunits, was activated after RyR-mediated Ca2+ release, suggesting a feedforward mechanism between NOX and RyR. Collectively, our data suggest a novel mechanism that is instrumental in sustaining physiological levels of ROS required for axonal growth of hippocampal neurons.

Rab35 Functions in Axon Elongation Are Regulated by P53-Related Protein Kinase in a Mechanism That Involves Rab35 Protein Degradation and the Microtubule-Associated Protein 1B.

UNLABELLED: Rab35 is a key protein for cargo loading in the recycling endosome. In neuronal immortalized cells, Rab35 promotes neurite differentiation. Here we describe that Rab35 favors axon elongation in rat primary neurons in an activity-dependent manner. In addition, we show that the p53-related protein kinase (PRPK) negatively regulates axonal elongation by reducing Rab35 protein levels through the ubiquitin-proteasome degradation pathway. PRPK-induced Rab35 degradation is regulated by its interaction with microtubule-associated protein 1B (MAP1B), a microtubule stabilizing binding protein essential for axon elongation. Consistently, axon defects found in MAP1B knock-out neurons were reversed by Rab35 overexpression or PRPK inactivation suggesting an epistatic relationship among these proteins. These results define a novel mechanism to support axonal elongation, by which MAP1B prevents PRPK-induced Rab35 degradation. Such a mechanism allows Rab35-mediated axonal elongation and connects the regulation of actin dynamics with membrane trafficking. In addition, our study reveals for the first time that the ubiquitin-proteasome degradation pathway regulates a Rab GTPase. SIGNIFICANCE STATEMENT: Rab35 is required for axonal outgrowth. We define that its protein levels are negatively regulated by p53-related protein kinase (PRPK). We show that microtubule-associated protein 1B (MAP1B) interacts with PRPK, preventing PRPK-dependent Rab35 proteasome degradation. We demonstrate that Rab35 regulates Cdc42 activity in neurons. This is the first evidence showing that a Rab protein is regulated by degradation dependent on the ubiquitin-proteasome system.

The NAV2 homolog Sickie regulates F-actin-mediated axonal growth in Drosophila mushroom body neurons via the non-canonical Rac-Cofilin pathway.

The Rac-Cofilin pathway is essential for cytoskeletal remodeling to control axonal development. Rac signals through the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth and through a Pak-independent non-canonical pathway to promote outgrowth. Whether this non-canonical pathway converges to promote Cofilin-dependent F-actin reorganization in axonal growth remains elusive. We demonstrate that Sickie, a homolog of the human microtubule-associated protein neuron navigator 2, cell-autonomously regulates axonal growth of Drosophila mushroom body (MB) neurons via the non-canonical pathway. Sickie was prominently expressed in the newborn F-actin-rich axons of MB neurons. A sickie mutant exhibited axonal growth defects, and its phenotypes were rescued by exogenous expression of Sickie. We observed phenotypic similarities and genetic interactions among sickie and Rac-Cofilin signaling components. Using the MARCM technique, distinct F-actin and phospho-Cofilin patterns were detected in developing axons mutant for sickie and Rac-Cofilin signaling regulators. The upregulation of Cofilin function alleviated the axonal defect of the sickie mutant. Epistasis analyses revealed that Sickie suppresses the LIMK overexpression phenotype and is required for Pak-independent Rac1 and Slingshot phosphatase to counteract LIMK. We propose that Sickie regulates F-actin-mediated axonal growth via the non-canonical Rac-Cofilin pathway in a Slingshot-dependent manner.

Axon position within the corpus callosum determines contralateral cortical projection.

How developing axons in the corpus callosum (CC) achieve their homotopic projection to the contralateral cortex remains unclear. We found that axonal position within the CC plays a critical role in this projection. Labeling of nearby callosal axons in mice showed that callosal axons were segregated in an orderly fashion, with those from more medial cerebral cortex located more dorsally and subsequently projecting to more medial contralateral cortical regions. The normal axonal order within the CC was grossly disturbed when semaphorin3A/neuropilin-1 signaling was disrupted. However, the order in which axons were positioned within the CC still determined their contralateral projection, causing a severe disruption of the homotopic contralateral projection that persisted at postnatal day 30, when the normal developmental refinement of contralateral projections is completed in wild-type (WT) mice. Thus, the orderly positioning of axons within the CC is a primary determinant of how homotopic interhemispheric projections form in the contralateral cortex.

MicroRNA-132 is enriched in developing axons, locally regulates Rasa1 mRNA, and promotes axon extension.

Developing axons can locally synthesize proteins, with roles in axon growth, guidance, and regeneration, but the mechanisms that regulate axonal mRNA translation are not well understood. MicroRNAs (miRNAs) are important regulators of translation but have still been little characterized in developing axons. Here we study mouse dorsal root ganglion (DRG) axons and show that their extension is impaired by conditional deficiency of the miRNA-processing enzyme Dicer in vitro and in vivo. A screen for axonal localization identifies a specific set of miRNAs preferentially enriched within the developing axon. High axonal expression and preferential localization were observed for miR-132, a miRNA previously known for roles in dendrites and dysregulation in major neurologic diseases. miR-132 knockdown reduced extension of cultured DRG axons, whereas overexpression increased extension. Mechanistically, miR-132 regulated the mRNA for the Ras GTPase activator Rasa1, a novel target in neuronal function. Moreover, miR-132 regulation of Rasa1 translation was seen in severed axons, demonstrating miRNA function locally within the axon. miR-132 expression in DRGs peaked in the period of maximum axon growth in vivo, consistent with its effect on axon growth, and suggesting a role as a developmental timer. Together, these findings identify miR-132 as a positive regulator of developing axon extension, acting through repression of Rasa1 mRNA, in a mechanism that operates locally within the axon.

In Vivo Time-Lapse Imaging in the Zebrafish Lateral Line: A Flexible, Open-Ended Research Project for an Undergraduate Neurobiology Laboratory Course.

The lateral line sensory system in fish detects movements in the water and allows fish to respond to predators, prey, and other stimuli. As the lateral line forms in the first two days of zebrafish development, axons extend caudally along the lateral surface of the fish, eventually forming synapses with hair cells of neuromasts. Growing lateral line axons are located superficially under the skin and can be labeled in living zebrafish using fluorescent protein expression. This system provides a relatively straightforward approach for in vivo time-lapse imaging of neuronal development in an undergraduate setting. Here we describe an upper-level neurobiology laboratory module in which students investigate aspects of axonal development in the zebrafish lateral line system. Students learn to handle and image living fish, collect time-lapse videos of moving mitochondria, and quantitatively measure mitochondrial dynamics by generating and analyzing kymographs of their movements. Energy demands may differ between axons with extending growth cones versus axons that have already reached their targets and are forming synapses. Since relatively little is known about this process in developing lateral line axons, students generate and test their own hypotheses regarding how mitochondrial dynamics may differ at two different time points in axonal development. Students also learn to incorporate into their analysis a powerful yet accessible quantitative tool, the kymograph, which is used to graph movement over time. After students measure and quantify dynamics in living fish at 1 and 2 days post fertilization, this module extends into independent projects, in which students can expand their studies in a number of different, inquiry-driven directions. The project can also be pared down for courses that wish to focus solely on the quantitative analysis (without fish handling), or vice versa. This research module provides a useful approach for the design of open-ended laboratory research projects that integrate the scientific process into undergraduate Biology courses, as encouraged by the AAAS and NSF Vision and Change Initiative.

Axon development is regulated at genetic and proteomic interfaces between the integrin adhesome and the RPM-1 ubiquitin ligase signaling hub.

Integrin signaling plays important roles in development and disease. An adhesion signaling network called the integrin adhesome has been principally defined using bioinformatics and proteomics. To date, the adhesome has not been studied using integrated proteomic and genetic approaches. Here, proteomic studies in C. elegans identified physical associations between the RPM-1 ubiquitin ligase signaling hub and numerous adhesome components including Talin, Kindlin and beta-integrin. C. elegans RPM-1 is orthologous to human MYCBP2, a prominent player in nervous system development associated with a neurodevelopmental disorder. Using neuron-specific, CRISPR loss-of-function strategies, we show that core adhesome components affect axon development and interact genetically with RPM-1. Mechanistically, Talin opposes RPM-1 in a functional 'tug-of-war' on growth cones that is required for accurate axon termination. Thus, our findings orthogonally validate the adhesome via multi-component genetic and physical interfaces with a key neuronal signaling hub and identify new links between the adhesome and brain disorders.

Inter-axonal molecular crosstalk via Lumican proteoglycan sculpts murine cervical corticospinal innervation by distinct subpopulations.

How CNS circuits sculpt their axonal arbors into spatially and functionally organized domains is not well understood. Segmental specificity of corticospinal connectivity is an exemplar for such regional specificity of many axon projections. Corticospinal neurons (CSN) innervate spinal and brainstem targets with segmental precision, controlling voluntary movement. Multiple molecularly distinct CSN subpopulations innervate the cervical cord for evolutionarily enhanced precision of forelimb movement. Evolutionarily newer CSNBC-lat exclusively innervate bulbar-cervical targets, while CSNmedial are heterogeneous; distinct subpopulations extend axons to either bulbar-cervical or thoraco-lumbar segments. We identify that Lumican controls balance of cervical innervation between CSNBC-lat and CSNmedial axons during development, which is maintained into maturity. Lumican, an extracellular proteoglycan expressed by CSNBC-lat, non-cell-autonomously suppresses cervical collateralization by multiple CSNmedial subpopulations. This inter-axonal molecular crosstalk between CSN subpopulations controls murine corticospinal circuitry refinement and forelimb dexterity. Such crosstalk is generalizable beyond the corticospinal system for evolutionary incorporation of new neuron populations into preexisting circuitry.

Transcriptional Analysis-Based Alterations Affecting Neuritogenesis of the Peripheral Nervous System in Psoriasis.

An increasing amount of evidence indicates the critical role of the cutaneous nervous system in the initiation and maintenance of psoriatic skin lesions by neurogenic inflammation. However, molecular mechanisms affecting cutaneous neurons are largely uncharacterized. Therefore, we reanalyzed a psoriatic RNA sequencing dataset from published transcriptome experiments of nearly 300 individuals. Using the Ingenuity Pathway Analysis software, we associated several hundreds of differentially expressed transcripts (DETs) to nervous system development and functions. Since neuronal projections were previously reported to be affected in psoriasis, we performed an in-depth analysis of neurite formation-related process. Our in silico analysis suggests that SEMA-PLXN and ROBO-DCC-UNC5 regulating axonal growth and repulsion are differentially affected in non-lesional and lesional skin samples. We identified opposing expressional alterations in secreted ligands for axonal guidance signaling (RTN4/NOGOA, NTNs, SEMAs, SLITs) and non-conventional axon guidance regulating ligands, including WNT5A and their receptors, modulating axon formation. These differences in neuritogenesis may explain the abnormal cutaneous nerve filament formation described in psoriatic skin. The processes also influence T-cell activation and infiltration, thus highlighting an additional angle of the crosstalk between the cutaneous nervous system and the immune responses in psoriasis pathogenesis, in addition to the known neurogenic pro-inflammatory mediators.

The ER ladder is a unique morphological feature of developing mammalian axons.

The endoplasmic reticulum (ER) confronts a challenge to accommodate long, smooth ER tubules into the structural complexity of the axonal compartment. Here, we describe a morphological feature for the axonal ER network in developing neurons we termed the ER ladder. Axonal ER ladders are composed of rungs that wrap tightly around the microtubule bundle and dynamic rails, which slide across microtubules. We found that the ER-shaping protein Reticulon 2 determines the architecture and dynamics of the axonal ER ladder by modulating its interaction with microtubules. Moreover, we show that ER ladder depletion impairs the trafficking of associated vesicular axonal cargoes. Finally, we demonstrate that stromal interaction molecule 1 (Stim1) localizes to ER rungs and translocates to ER-plasma membrane contact sites upon depletion of luminal Ca2+. Our findings uncover fundamental insights into the structural and functional organization of the axonal ER network in developing mammalian neurons.

Pum2 Shapes the Transcriptome in Developing Axons through Retention of Target mRNAs in the Cell Body.

Localized protein synthesis is fundamental for neuronal development, maintenance, and function. Transcriptomes in axons and soma are distinct, but the mechanisms governing the composition of axonal transcriptomes and their developmental regulation are only partially understood. We found that the binding motif for the RNA-binding proteins Pumilio 1 and 2 (Pum1 and Pum2) is underrepresented in transcriptomes of developing axons. Introduction of Pumilio-binding elements (PBEs) into mRNAs containing a ß-actin zipcode prevented axonal localization and translation. Pum2 is restricted to the soma of developing neurons, and Pum2 knockdown or blocking its binding to mRNA caused the appearance and translation of PBE-containing mRNAs in axons. Pum2-deficient neurons exhibited axonal growth and branching defects in vivo and impaired axon regeneration in vitro. These results reveal that Pum2 shapes axonal transcriptomes by preventing the transport of PBE-containing mRNAs into axons, and they identify somatic mRNAs retention as a mechanism for the temporal control of intra-axonal protein synthesis.

The Microtubule Regulator NEK7 Coordinates the Wiring of Cortical Parvalbumin Interneurons.

Functional networks in the mammalian cerebral cortex rely on the interaction between glutamatergic pyramidal cells and GABAergic interneurons. Both neuronal populations exhibit an extraordinary divergence in morphology and targeting areas, which ultimately dictate their precise function in cortical circuits. How these prominent morphological differences arise during development is not well understood. Here, we conducted a high-throughput screen for genes differentially expressed by pyramidal cells and interneurons during cortical wiring. We found that NEK7, a kinase involved in microtubule polymerization, is mostly expressed in parvalbumin (PV+) interneurons at the time when they establish their connectivity. Functional experiments revealed that NEK7-deficient PV+ interneurons show altered microtubule dynamics, axon growth cone steering and reduced axon length, arbor complexity, and total number of synaptic contacts formed with pyramidal cells. Altogether, our results reveal a molecular mechanism by which the microtubule-associated kinase NEK7 regulates the wiring of PV+ interneurons.

Axonal microRNAs: localization, function and regulatory mechanism during axon development.

Subcellular localization and translation of messenger RNAs are essential for the regulation of neuronal development and synaptic function. As post-transcriptional regulators, microRNAs (miRNAs) have been emerging as central players in the development and maturation of the nervous system. Recent discoveries reveal the critical functions of miRNAs in the axon of neurons via multiple pathways of molecular regulation. Here, we introduce methods for isolating axonal miRNAs and review recent findings on the localization and function as well as regulatory mechanism of axonal miRNAs during axon development.

Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development.

During mammalian embryonic development, neurons polarize to create distinct cellular compartments of axon and dendrite that inherently differ in form and function, providing the foundation for directional signaling in the nervous system. Polarization results from spatio-temporal segregation of specific proteins' activities to discrete regions of the neuron to dictate axonal vs. dendritic fate. We aim to manipulate axon formation by directed subcellular localization of crucial intracellular protein function. Here we report critical steps toward the development of a nanotechnology for localized subcellular introduction and retention of an intracellular kinase, LKB1, crucial regulator of axon formation. This nanotechnology will spatially manipulate LKB1-linked biomagnetic nanocomplexes (LKB1-NCs) in developing rodent neurons in culture and in vivo. We created a supramolecular assembly for LKB1 rapid neuronal uptake and prolonged cytoplasmic stability. LKB1-NCs retained kinase activity and phosphorylated downstream targets. NCs were successfully delivered to cultured embryonic hippocampal neurons, and were stable in the cytoplasm for 2 days, sufficient time for axon formation. Importantly, LKB1-NCs promoted axon formation in these neurons, representing unique proof of concept for the sufficiency of intracellular protein function in dictating a central developmental event. Lastly, we established NC delivery into cortical progenitors in live rat embryonic brain in utero. Our nanotechnology provides a viable platform for spatial manipulation of intracellular protein-activity, to dictate central events during neuronal development.