Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.

LTK and ALK promote neuronal polarity and cortical migration by inhibiting IGF1R activity.

The establishment of axon-dendrite polarity is fundamental for radial migration of neurons, cortical patterning, and formation of neuronal circuits. Here, we show that the receptor tyrosine kinases, Ltk and Alk, are required for proper neuronal polarization. In isolated primary mouse embryonic neurons, the loss of Ltk and/or Alk causes a multiple axon phenotype. In mouse embryos and newborn pups, the absence of Ltk and Alk delays neuronal migration and subsequent cortical patterning. In adult cortices, neurons with aberrant neuronal projections are evident and axon tracts in the corpus callosum are disrupted. Mechanistically, we show that the loss of Alk and Ltk increases the cell-surface expression and activity of the insulin-like growth factor 1 receptor (Igf-1r), which activates downstream PI3 kinase signaling to drive the excess axon phenotype. Our data reveal Ltk and Alk as new regulators of neuronal polarity and migration whose disruption results in behavioral abnormalities.

KIF2A deficiency causes early-onset neurodegeneration.

KIF2A is an atypical kinesin that has the capacity to depolymerize microtubules. Patients carrying mutations in KIF2A suffer from progressive microcephaly and mental disabilities. While the role of this protein is well documented in neuronal migration, the relationship between its dysfunction and the pathobiology of brain disorders is unclear. Here, we report that KIF2A is dispensable for embryogenic neurogenesis but critical in postnatal stages for maturation, connectivity, and maintenance of neurons. We used a conditional approach to inactivate KIF2A in cortical progenitors, nascent postmitotic neurons, and mature neurons in mice. We show that the lack of KIF2A alters microtubule dynamics and disrupts several microtubule-dependent processes, including neuronal polarity, neuritogenesis, synaptogenesis, and axonal transport. KIF2A-deficient neurons exhibit aberrant electrophysiological characteristics, neuronal connectivity, and function, leading to their loss. The role of KIF2A is not limited to development, as fully mature neurons require KIF2A for survival. Our results emphasize an additional function of KIF2A and help explain how its mutations lead to brain disorders.

A Role for Second Messengers in Axodendritic Neuronal Polarity.

Neuronal polarization is a complex molecular process regulated by intrinsic and extrinsic mechanisms. Nerve cells integrate multiple extracellular cues to generate intracellular messengers that ultimately control cell morphology, metabolism, and gene expression. Therefore, second messengers' local concentration and temporal regulation are crucial elements for acquiring a polarized morphology in neurons. This review article summarizes the main findings and current understanding of how Ca2+, IP3, cAMP, cGMP, and hydrogen peroxide control different aspects of neuronal polarization, and highlights questions that still need to be resolved to fully understand the fascinating cellular processes involved in axodendritic polarization.

Inheritance and flexibility of cell polarity: a clue for understanding human brain development and evolution.

Cell polarity is fundamentally important for understanding brain development. Here, we hypothesize that the inheritance and flexibility of cell polarity during neocortex development could be implicated in neocortical evolutionary expansion. Molecular and morphological features of cell polarity may be inherited from one type of progenitor cell to the other and finally transmitted to neurons. Furthermore, key cell types, such as basal progenitors and neurons, exhibit a highly flexible polarity. We suggest that both inheritance and flexibility of cell polarity are implicated in the amplification of basal progenitors and tangential dispersion of neurons, which are key features of the evolutionary expansion of the neocortex.

Microtubule polarity is instructive for many aspects of neuronal polarity.

Many neurons in bilaterian animals are polarized with functionally distinct axons and dendrites. Microtubule polarity, microtubule stability, and the axon initial segment (AIS) have all been shown to influence polarized transport in neurons. Each of these cytoskeletal cues could act independently to control axon and dendrite identity, or there could be a hierarchy in which one acts upstream of the others. Here we test the hypothesis that microtubule polarity acts as a master regulator of neuronal polarity by using a Drosophila genetic background in which some dendrites have normal minus-end-out microtubule polarity and others have the axonal plus-end-out polarity. In these mosaic dendrite arbors, we found that ribosomes, which are more abundant in dendrites than axons, were reduced in plus-end-out dendrites, while an axonal cargo was increased. In addition, we determined that microtubule stability was different in plus-end-out and minus-end-out dendrites, with plus-end-out ones having more stable microtubules like axons. Similarly, we found that ectopic diffusion barriers, like those at the AIS, formed at the base of dendrites with plus-end-out regions. Thus, changes in microtubule polarity were sufficient to rearrange other cytoskeletal features associated with neuronal polarization. However, overall neuron shape was maintained with only subtle changes in branching in mosaic arbors. We conclude that microtubule polarity can act upstream of many aspects of intracellular neuronal polarization, but shape is relatively resilient to changes in microtubule polarity in vivo.

Crystal structure of Ankyrin-G in complex with a fragment of Neurofascin reveals binding mechanisms required for integrity of the axon initial segment.

The axon initial segment (AIS) has characteristically dense clustering of voltage-gated sodium channels (Nav), cell adhesion molecule Neurofascin 186 (Nfasc), and neuronal scaffold protein Ankyrin-G (AnkG) in neurons, which facilitates generation of an action potential and maintenance of axonal polarity. However, the mechanisms underlying AIS assembly, maintenance, and plasticity remain poorly understood. Here, we report the high-resolution crystal structure of the AnkG ankyrin repeat (ANK repeat) domain in complex with its binding site in the Nfasc cytoplasmic tail that shows, in conjunction with binding affinity assays with serial truncation variants, the molecular basis of AnkG-Nfasc binding. We confirm AnkG interacts with the FIGQY motif in Nfasc, and we identify another region required for their high affinity binding. Our structural analysis revealed that ANK repeats form 4 hydrophobic or hydrophilic layers in the AnkG inner groove that coordinate interactions with essential Nfasc residues, including F1202, E1204, and Y1212. Moreover, we show disruption of the AnkG-Nfasc complex abolishes Nfasc enrichment at the AIS in cultured mouse hippocampal neurons. Finally, our structural and biochemical analysis indicated that L1 syndrome-associated mutations in L1CAM, a member of the L1 immunoglobulin family proteins including Nfasc, L1CAM, NrCAM, and CHL1, compromise binding with ankyrins. Taken together, these results define the mechanisms underlying AnkG-Nfasc complex formation and show that AnkG-dependent clustering of Nfasc is required for AIS integrity.

Ciliated sensory neurons can regenerate axons after complete axon removal.

Axon regeneration helps maintain lifelong function of neurons in many animals. Depending on the site of injury, new axons can grow either from the axon stump (after distal injury) or from the tip of a dendrite (after proximal injury). However, some neuron types do not have dendrites to be converted to a regenerating axon after proximal injury. For example, many sensory neurons receive information from a specialized sensory cilium rather than a branched dendrite arbor. We hypothesized that the lack of traditional dendrites would limit the ability of ciliated sensory neurons to respond to proximal axon injury. We tested this hypothesis by performing laser microsurgery on ciliated lch1 neurons in Drosophila larvae and tracking cells over time. These cells survived proximal axon injury as well as distal axon injury, and, like many other neurons, initiated growth from the axon stump after distal injury. After proximal injury, neurites regrew in a surprisingly flexible manner. Most cells initiated outgrowth directly from the cell body, but neurite growth could also emerge from the short axon stump or base of the cilium. New neurites were often branched. Although outgrowth after proximal axotomy was variable, it depended on the core DLK axon injury signaling pathway. Moreover, each cell had at least one new neurite specified as an axon based on microtubule polarity and accumulation of the endoplasmic reticulum. We conclude that ciliated sensory neurons are not intrinsically limited in their ability to grow a new axon after proximal axon removal.

GPR3 accelerates neurite outgrowth and neuronal polarity formation via PI3 kinase-mediating signaling pathway in cultured primary neurons.

During neuronal development, immature neurons extend neurites and subsequently polarize to form an axon and dendrites. We have previously reported that G protein-coupled receptor 3 (GPR3) levels increase during neuronal development, and that GPR3 has functions in neurite outgrowth and neuronal differentiation in cerebellar granular neurons. Moreover, GPR3 is transported and concentrated at the tips of neurite, thereby contributing to the local activation of protein kinase A (PKA). However, the signaling pathways for GPR3-mediated neurite outgrowth and its subsequent effects on neuronal polarization have not yet been elucidated. We therefore analyzed the signaling pathways related to GPR3-mediated neurite outgrowth, and also focused on the possible roles of GPR3 in axon polarization. We demonstrated that, in cerebellar granular neurons, GPR3-mediated neurite outgrowth was mediated by multiple signaling pathways, including those of PKA, extracellular signal-regulated kinases (ERKs), and most strongly phosphatidylinositol 3-kinase (PI3K). In addition, the GPR3-mediated activation of neurite outgrowth was associated with G protein-coupled receptor kinase 2 (GRK2)-mediated signaling and phosphorylation of the C-terminus serine/threonine residues of GPR3, which affected downstream protein kinase B (Akt) signaling. We further demonstrated that GPR3 was transiently increased early in the development of rodent hippocampal neurons. It was subsequently concentrated at the tip of the longest neurite, and was thus associated with accelerated polarity formation in a PI3K-dependent manner in rat hippocampal neurons. In addition, GPR3 knockout in mouse hippocampal neurons led to delayed neuronal polarity formation, thereby affecting the dephosphorylation of collapsing response mediator protein 2 (CRMP2), which is downstream of the PI3K signaling pathway. Taken together, these findings suggest that the intrinsic expression of GPR3 in differentiated neurons constitutively activates PI3K-mediated signaling pathway predominantly, thus accelerating neurite outgrowth and further augmenting polarity formation in primary cultured neurons.

CAMSAP1 breaks the homeostatic microtubule network to instruct neuronal polarity.

The establishment of axon/dendrite polarity is fundamental for neurons to integrate into functional circuits, and this process is critically dependent on microtubules (MTs). In the early stages of the establishment process, MTs in axons change dramatically with the morphological building of neurons; however, how the MT network changes are triggered is unclear. Here we show that CAMSAP1 plays a decisive role in the neuronal axon identification process by regulating the number of MTs. Neurons lacking CAMSAP1 form a multiple axon phenotype in vitro, while the multipolar-bipolar transition and radial migration are blocked in vivo. We demonstrate that the polarity regulator MARK2 kinase phosphorylates CAMSAP1 and affects its ability to bind to MTs, which in turn changes the protection of MT minus-ends and also triggers asymmetric distribution of MTs. Our results indicate that the polarized MT network in neurons is a decisive factor in establishing axon/dendritic polarity and is initially triggered by polarized signals.

Deciphering the Tubulin Language: Molecular Determinants and Readout Mechanisms of the Tubulin Code in Neurons.

Microtubules (MTs) are dynamic components of the cell cytoskeleton involved in several cellular functions, such as structural support, migration and intracellular trafficking. Despite their high similarity, MTs have functional heterogeneity that is generated by the incorporation into the MT lattice of different tubulin gene products and by their post-translational modifications (PTMs). Such regulations, besides modulating the tubulin composition of MTs, create on their surface a "biochemical code" that is translated, through the action of protein effectors, into specific MT-based functions. This code, known as "tubulin code", plays an important role in neuronal cells, whose highly specialized morphologies and activities depend on the correct functioning of the MT cytoskeleton and on its interplay with a myriad of MT-interacting proteins. In recent years, a growing number of mutations in genes encoding for tubulins, MT-interacting proteins and enzymes that post-translationally modify MTs, which are the main players of the tubulin code, have been linked to neurodegenerative processes or abnormalities in neural migration, differentiation and connectivity. Nevertheless, the exact molecular mechanisms through which the cell writes and, downstream, MT-interacting proteins decipher the tubulin code are still largely uncharted. The purpose of this review is to describe the molecular determinants and the readout mechanisms of the tubulin code, and briefly elucidate how they coordinate MT behavior during critical neuronal events, such as neuron migration, maturation and axonal transport.

A novel labeling strategy reveals that myosin Va and myosin Vb bind the same dendritically polarized vesicle population.

Neurons are specialized cells with a polarized geometry and several distinct subdomains that require specific complements of proteins. Delivery of transmembrane proteins requires vesicle transport, which is mediated by molecular motor proteins. The myosin V family of motor proteins mediates transport to the barbed end of actin filaments, and little is known about the vesicles bound by myosin V in neurons. We developed a novel strategy to visualize myosin V-labeled vesicles in cultured hippocampal neurons and systematically characterized the vesicle populations labeled by myosin Va and Vb. We find that both myosins bind vesicles that are polarized to the somatodendritic domain where they undergo bidirectional long-range transport. A series of two-color imaging experiments showed that myosin V specifically colocalized with two different vesicle populations: vesicles labeled with the transferrin receptor and vesicles labeled by low-density lipoprotein receptor. Finally, coexpression with Kinesin-3 family members found that myosin V binds vesicles concurrently with KIF13A or KIF13B, supporting the hypothesis that coregulation of kinesins and myosin V on vesicles is likely to play an important role in neuronal vesicle transport. We anticipate that this new assay will be applicable in a broad range of cell types to determine the function of myosin V motor proteins.

Tuba Activates Cdc42 during Neuronal Polarization Downstream of the Small GTPase Rab8a.

The acquisition of neuronal polarity is a complex molecular process that depends on changes in cytoskeletal dynamics and directed membrane traffic, regulated by the Rho and Rab families of small GTPases, respectively. However, during axon specification, a molecular link that couples these protein families has yet to be identified. In this paper, we describe a new positive feedback loop between Rab8a and Cdc42, coupled by Tuba, a Cdc42-specific guanine nucleotide-exchange factor (GEF), that ensures a single axon generation in rodent hippocampal neurons from embryos of either sex. Accordingly, Rab8a or Tuba gain-of-function generates neurons with supernumerary axons whereas Rab8a or Tuba loss-of-function abrogated axon specification, phenocopying the well-established effect of Cdc42 on neuronal polarity. Although Rab8 and Tuba do not interact physically, the activity of Rab8 is essential to generate a proximal to distal axonal gradient of Tuba in cultured neurons. Tuba-associated and Rab8a-associated polarity defects are also evidenced in vivo, since dominant negative (DN) Rab8a or Tuba knock-down impairs cortical neuronal migration in mice. Our results suggest that Tuba coordinates directed vesicular traffic and cytoskeleton dynamics during neuronal polarization.SIGNIFICANCE STATEMENT The morphologic, biochemical, and functional differences observed between axon and dendrites, require dramatic structural changes. The extension of an axon that is 1 µm in diameter and grows at rates of up to 500 µm/d, demands the confluence of two cellular processes: directed membrane traffic and fine-tuned cytoskeletal dynamics. In this study, we show that both processes are integrated in a positive feedback loop, mediated by the guanine nucleotide-exchange factor (GEF) Tuba. Tuba connects the activities of the Rab GTPase Rab8a and the Rho GTPase Cdc42, ensuring the generation of a single axon in cultured hippocampal neurons and controlling the migration of cortical neurons in the developing brain. Finally, we provide compelling evidence that Tuba is the GEF that mediates Cdc42 activation during the development of neuronal polarity.

Microtubule organization of vertebrate sensory neurons in vivo.

Dorsal root ganglion (DRG) neurons are the predominant cell type that innervates the vertebrate skin. They are typically described as pseudounipolar cells that have central and peripheral axons branching from a single root exiting the cell body. The peripheral axon travels within a nerve to the skin, where free sensory endings can emerge and branch into an arbor that receives and integrates information. In some immature vertebrates, DRG neurons are preceded by Rohon-Beard (RB) neurons. While the sensory endings of RB and DRG neurons function like dendrites, we use live imaging in zebrafish to show that they have axonal plus-end-out microtubule polarity at all stages of maturity. Moreover, we show both cell types have central and peripheral axons with plus-end-out polarity. Surprisingly, in DRG neurons these emerge separately from the cell body, and most cells never acquire the signature pseudounipolar morphology. Like another recently characterized cell type that has multiple plus-end-out neurites, ganglion cells in Nematostella, RB and DRG neurons maintain a somatic microtubule organizing center even when mature. In summary, we characterize key cellular and subcellular features of vertebrate sensory neurons as a foundation for understanding their function and maintenance.

Sec3 exocyst component knockdown inhibits axonal formation and cortical neuronal migration during brain cortex development.

Neurons are the largest known cells, with complex and highly polarized morphologies and consist of a cell body (soma), several dendrites, and a single axon. The establishment of polarity necessitates initial axonal outgrowth in concomitance with the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles primarily at the neuronal growth cone membrane. The multiprotein exocyst complex drives spatial location and specificity of vesicle fusion at plasma membrane. However, the specific participation of its different proteins on neuronal differentiation has not been fully established. In the present work we analyzed the role of Sec3, a prominent exocyst complex protein on neuronal differentiation. Using mice hippocampal primary cultures, we determined that Sec3 is expressed in neurons at early stages prior to neuronal polarization. Furthermore, we determined that silencing of Sec3 in mice hippocampal neurons in culture precluded polarization. Moreover, using in utero electroporation experiments, we determined that Sec3 knockdown affected cortical neurons migration and morphology during neocortex formation. Our results demonstrate that the exocyst complex protein Sec3 plays an important role in axon formation in neuronal differentiation and the migration of neuronal progenitors during cortex development.

Adenosine A2A Receptors Contribute to the Radial Migration of Cortical Projection Neurons through the Regulation of Neuronal Polarization and Axon Formation.

Cortical interneurons born in the subpallium reach the cortex through tangential migration, whereas pyramidal cells reach their final position by radial migration. Purinergic signaling via P2Y1 receptors controls the migration of intermediate precursor cells from the ventricular zone to the subventricular zone. It was also reported that the blockade of A2A receptors (A2AR) controls the tangential migration of somatostatin+ interneurons. Here we found that A2AR control radial migration of cortical projection neurons. In A2AR-knockout (KO) mouse embryos or naïve mouse embryos exposed to an A2AR antagonist, we observed an accumulation of early-born migrating neurons in the lower intermediate zone at late embryogenesis. In utero knockdown of A2AR also caused an accumulation of neurons at the lower intermediate zone before birth. This entails the presently identified ability of A2AR to promote multipolar-bipolar transition and axon formation, critical for the transition of migrating neurons from the intermediate zone to the cortical plate. This effect seems to require extracellular ATP-derived adenosine since a similar accumulation of neurons at the lower intermediate zone was observed in mice lacking ecto-5'-nucleotidase (CD73-KO). These findings frame adenosine as a fine-tune regulator of the wiring of cortical inhibitory and excitatory networks.

Regulation of Caenorhabditis elegans neuronal polarity by heterochronic genes.

Many neurons display characteristic patterns of synaptic connections that are under genetic control. The Caenorhabditis elegans DA cholinergic motor neurons form synaptic connections only on their dorsal axons. We explored the genetic pathways that specify this polarity by screening for gene inactivations and mutations that disrupt this normal polarity of a DA motorneuron. A RAB-3::GFP fusion protein that is normally localized to presynaptic terminals along the dorsal axon of the DA9 motorneuron was used to screen for gene inactivations that disrupt the DA9 motorneuron polarity. This screen identified heterochronic genes as major regulators of DA neuron presynaptic polarity. In many heterochronic mutants, presynapses of this cholinergic motoneuron are mislocalized to the dendrite at the ventral side: inactivation of the blmp-1 transcription factor gene, the lin-29/Zn finger transcription factor, lin-28/RNA binding protein, and the let-7miRNA gene all disrupt the presynaptic polarity of this DA cholinergic neuron. We also show that the dre-1/F box heterochronic gene functions early in development to control maintenance of polarity at later stages, and that a mutation in the let-7 heterochronic miRNA gene causes dendritic misplacement of RAB-3 presynaptic markers that colocalize with muscle postsynaptic terminals ectopically. We propose that heterochronic genes are components in the UNC-6/Netrin pathway of synaptic polarity of these neurons. These findings highlight the role of heterochronic genes in postmitotic neuronal patterning events.

Neurons survive simultaneous injury to axons and dendrites and regrow both types of processes in vivo.

Neurons extend dendrites and axons to receive and send signals. If either type of process is removed, the cell cannot function. Rather than undergoing cell death, some neurons can regrow axons and dendrites. Axon and dendrite regeneration have been examined separately and require sensing the injury and reinitiating the correct growth program. Whether neurons in vivo can sense and respond to simultaneous axon and dendrite injury with polarized regeneration has not been explored. To investigate the outcome of simultaneous axon and dendrite damage, we used a Drosophila model system in which neuronal polarity, axon regeneration, and dendrite regeneration have been characterized. After removal of the axon and all but one dendrite, the remaining dendrite was converted to a process that had a long unbranched region that extended over long distances and a region where shorter branched processes were added. These observations suggested axons and dendrites could regrow at the same time. To further test the capacity of neurons to implement polarized regeneration after axon and dendrite damage, we removed all neurites from mature neurons. In this case a long unbranched neurite and short branched neurites were regrown from the stripped cell body. Moreover, the long neurite had axonal plus-end-out microtubule polarity and the shorter neurites had mixed polarity consistent with dendrite identity. The long process also accumulated endoplasmic reticulum at its tip like regenerating axons. We conclude that neurons in vivo can respond to simultaneous axon and dendrite injury by initiating growth of a new axon and new dendrites.

Perturbation of canonical and non-canonical BMP signaling affects migration, polarity and dendritogenesis of mouse cortical neurons.

Bone morphogenetic protein (BMP) signaling has been implicated in the regulation of patterning of the forebrain and as a regulator of neurogenesis and gliogenesis in the mammalian cortex. However, its role in other aspects of cortical development in vivo remains unexplored. We hypothesized that BMP signaling might regulate additional processes during the development of cortical neurons after observing active BMP signaling in a spatiotemporally dynamic pattern in the mouse cortex. Our investigation revealed that BMP signaling specifically regulates the migration, polarity and the dendritic morphology of upper layer cortical neurons born at E15.5. On further dissection of the role of canonical and non-canonical BMP signaling in each of these processes, we found that migration of these neurons is regulated by both pathways. Their polarity, however, appears to be affected more strongly by canonical BMP signaling, whereas dendritic branch formation appears to be somewhat more strongly affected by LIMK-mediated non-canonical BMP signaling.

CAMSAP3 maintains neuronal polarity through regulation of microtubule stability.

The molecular mechanisms that guide each neuron to become polarized, forming a single axon and multiple dendrites, remain unknown. Here we show that CAMSAP3 (calmodulin-regulated spectrin-associated protein 3), a protein that regulates the minus-end dynamics of microtubules, plays a key role in maintaining neuronal polarity. In mouse hippocampal neurons, CAMSAP3 was enriched in axons. Although axonal microtubules were generally acetylated, CAMSAP3 was preferentially localized along a less-acetylated fraction of the microtubules. CAMSAP3-mutated neurons often exhibited supernumerary axons, along with an increased number of neurites having nocodazole-resistant/acetylated microtubules compared with wild-type neurons. Analysis using cell lines showed that CAMSAP3 depletion promoted tubulin acetylation, and conversely, mild overexpression of CAMSAP3 inhibited it, suggesting that CAMSAP3 works to retain nonacetylated microtubules. In contrast, CAMSAP2, a protein related to CAMSAP3, was detected along all neurites, and its loss did not affect neuronal polarity, nor did it cause increased tubulin acetylation. Depletion of α-tubulin acetyltransferase-1 (αTAT1), the key enzyme for tubulin acetylation, abolished CAMSAP3 loss-dependent multiple-axon formation. These observations suggest that CAMSAP3 sustains a nonacetylated pool of microtubules in axons, interfering with the action of αTAT1, and this process is important to maintain neuronal polarity.