Axonal Polarity Research Articles

Depletion of Inositol Polyphosphate 4-Phosphatase II Suppresses Callosal Axon Formation in the Developing Mice.

The corpus callosum is a bundle of nerve fibers that connects the two cerebral hemispheres and is essential for coordinated transmission of information between them. Disruption of early stages of callosal development can cause agenesis of the corpus callosum (AgCC), including both complete and partial callosal absence, causing mild to severe cognitive impairment. Despite extensive studies, the etiology of AgCC remains to be clarified due to the complicated mechanism involved in generating AgCC. The biological function of PI3K signaling including phosphatidylinositol-3,4,5-trisphosphate is well established in diverse biochemical processes including axon and dendrite morphogenesis, but the function of the closely related phosphatidylinositol-3,4,-bisphosphate (PI(3,4)P2) signaling, particularly in the nervous system, is largely unknown. Here, we provide the first report on the role of inositol polyphosphate 4-phosphatase II (INPP4B), a PI(3,4)P2 metabolizing 4-phosphatase in the regulation of callosal axon formation. Depleting INPP4B by in utero electroporation suppressed medially directed callosal axon formation. Moreover, depletion of INPP4B significantly attenuated formation of Satb2-positive pyramidal neurons and axon polarization in cortical neurons during cortical development. Taken together, these data suggest that INPP4B plays a role in the regulating callosal axon formation by controlling axon polarization and the Satb2-positive pyramidal neuron population. Dysregulation of INPP4B during cortical development may be implicated in the generation of partial AgCC.

Frizzled3 Controls Axonal Polarity and Intermediate Target Entry during Striatal Pathway Development.

Striatal axon pathways mediate complex physiological functions and are an important therapeutic target, underscoring the need to define how these connections are established. Remarkably, the molecular programs regulating striatal pathway development remain poorly characterized. Here, we determine the embryonic ontogeny of the two main striatal pathways (striatonigral and striatopallidal) and identify novel (non)cell-autonomous roles for the axon guidance receptor Frizzled3 in uncharacterized aspects of striatal pathway formation (i.e., anterior-posterior axon guidance in the striatum and axon entry into the globus pallidus). Further, our results link Frizzled3 to corridor guidepost cell development and suggest that an abnormal distribution of these cells has unexpected, widespread effects on the development of different axon tracts (i.e., striatal and thalamocortical axons).

Early-stage development of human induced pluripotent stem cell-derived neurons.

Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Differentiated neurons from hiPSCs are expected to be useful for developing novel methods of treatment for various neurological diseases. However, the detailed process of functional maturation of hiPSC‐derived neurons (hiPS neurons) remains poorly understood. This study analyzes development of hiPS neurons, focusing specifically on early developmental stages through 48 hr after cell seeding; development was compared with that of primary cultured neurons derived from the rat hippocampus. At 5 hr after cell seeding, neurite formation occurs in a similar manner in both neuronal populations. However, very few neurons with axonal polarization were observed in the hiPS neurons even after 48 hr, indicating that hiPS neurons differentiate more slowly than rat neurons. We further investigated the elongation speed of axons and found that hiPS neuronal axons were slower. In addition, we characterized the growth cones. The localization patterns of skeletal proteins F‐actin, microtubule, and drebrin were similar to those of rat neurons, and actin depolymerization by cytochalasin D induced similar changes in cytoskeletal distribution in the growth cones between hiPS neurons and rat neurons. These results indicate that, during the very early developmental stage, hiPS neurons develop comparably to rat hippocampal neurons with regard to axonal differentiation, but the growth of axons is slower. © 2015 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Giant ankyrin-G: A critical innovation in vertebrate evolution of fast and integrated neuronal signaling

Axon initial segments (AISs) and nodes of Ranvier are sites of clustering of voltage-gated sodium channels (VGSCs) in nervous systems of jawed vertebrates that facilitate fast long-distance electrical signaling. We demonstrate that proximal axonal polarity as well as assembly of the AIS and normal morphogenesis of nodes of Ranvier all require a heretofore uncharacterized alternatively spliced giant exon of ankyrin-G (AnkG). This exon has sequence similarity to I-connectin/Titin and was acquired after the first round of whole-genome duplication by the ancestral ANK2/ANK3 gene in early vertebrates before development of myelin. The giant exon resulted in a new nervous system-specific 480-kDa polypeptide combining previously known features of ANK repeats and β-spectrin-binding activity with a fibrous domain nearly 150 nm in length. We elucidate previously undescribed functions for giant AnkG, including recruitment of β4 spectrin to the AIS that likely is regulated by phosphorylation, and demonstrate that 480-kDa AnkG is a major component of the AIS membrane "undercoat' imaged by platinum replica electron microscopy. Surprisingly, giant AnkG-knockout neurons completely lacking known AIS components still retain distal axonal polarity and generate action potentials (APs), although with abnormal frequency. Giant AnkG-deficient mice live to weaning and provide a rationale for survival of humans with severe cognitive dysfunction bearing a truncating mutation in the giant exon. The giant exon of AnkG is required for assembly of the AIS and nodes of Ranvier and was a transformative innovation in evolution of the vertebrate nervous system that now is a potential target in neurodevelopmental disorders.

Kalirin-9 and Kalirin-12 Play Essential Roles in Dendritic Outgrowth and Branching.

Proteins derived from the Kalrn gene, encoding 2 Rho guanine nucleotide exchange factor (GEF) domains, affect dendritic and axonal morphogenesis. The roles of endogenous Kalirin-9 (Kal9) and Kalirin-12 (Kal12), the Kalrn isoforms expressed before synaptogenesis, have not been studied in neurite growth and maturation during early development. The Caenorhabditis elegans and Drosophila melanogaster orthologues of Kalrn encode proteins equivalent to Kal9 but, lacking a kinase domain, neither organism expresses a protein equivalent to Kal12. Both in vivo and in vitro analyses of cortical neurons from total Kalrn knockout mice, lacking all major Kalirin isoforms, revealed a simplified dendritic arbor and reduced neurite length. Using isoform-specific shRNAs to reduce Kal9 or Kal12 expression in hippocampal cultures resulted in stunted dendritic outgrowth and branching in vitro, without affecting axonal polarity. Exposing hippocampal cultures to inhibitors of the first GEF domain of Kalirin (ITX3, Z62954982) blunted neurite outgrowth and branching, confirming its essential role, without altering the morphology of neurons not expressing Kalrn. In addition, exogenous expression of the active kinase domain unique to Kal12 increased neurite number and length, whereas that of the inactive kinase domain decreased neurite growth. Our results demonstrate that both endogenous Kal9 and endogenous Kal12 contribute to dendritic maturation in early development.

The value and cost of complexity in predictive modelling: role of tissue anisotropic conductivity and fibre tracts in neuromodulation

Objectives. Computational methods are increasingly used to optimize transcranial direct current stimulation (tDCS) dose strategies and yet complexities of existing approaches limit their clinical access. Since predictive modelling indicates the relevance of subject/pathology based data and hence the need for subject specific modelling, the incremental clinical value of increasingly complex modelling methods must be balanced against the computational and clinical time and costs. For example, the incorporation of multiple tissue layers and measured diffusion tensor (DTI) based conductivity estimates increase model precision but at the cost of clinical and computational resources. Costs related to such complexities aggregate when considering individual optimization and the myriad of potential montages. Here, rather than considering if additional details change current-flow prediction, we consider when added complexities influence clinical decisions. Approach. Towards developing quantitative and qualitative metrics of value/cost associated with computational model complexity, we considered field distributions generated by two 4 × 1 high-definition montages (m1 = 4 × 1 HD montage with anode at C3 and m2 = 4 × 1 HD montage with anode at C1) and a single conventional (m3 = C3-Fp2) tDCS electrode montage. We evaluated statistical methods, including residual error (RE) and relative difference measure (RDM), to consider the clinical impact and utility of increased complexities, namely the influence of skull, muscle and brain anisotropic conductivities in a volume conductor model. Main results. Anisotropy modulated current-flow in a montage and region dependent manner. However, significant statistical changes, produced within montage by anisotropy, did not change qualitative peak and topographic comparisons across montages. Thus for the examples analysed, clinical decision on which dose to select would not be altered by the omission of anisotropic brain conductivity. Significance. Results illustrate the need to rationally balance the role of model complexity, such as anisotropy in detailed current flow analysis versus value in clinical dose design. However, when extending our analysis to include axonal polarization, the results provide presumably clinically meaningful information. Hence the importance of model complexity may be more relevant with cellular level predictions of neuromodulation.

Role of PPAR γ in the Differentiation and Function of Neurons.

Neuronal processes (neurites and axons) have an important role in brain cells communication and, generally, they are damaged in neurodegenerative diseases. Recent evidence has showed that the activation of PPARγ pathway promoted neuronal differentiation and axon polarity. In addition, activation of PPARγ using thiazolidinediones (TZDs) prevented neurodegeneration by reducing neuronal death, improving mitochondrial function, and decreasing neuroinflammation in neuropathic pain. In this review, we will discuss important evidence that supports a possible role of PPARγ in neuronal development, improvement of neuronal health, and pain signaling. Therefore, activation of PPARγ is a potential target with therapeutic applications against neurodegenerative disorders, brain injury, and pain regulation.

Small-molecule axon-polarization studies enabled by a shear-free microfluidic gradient generator.

A deep understanding of the mechanisms behind neurite polarization and axon path-finding is important for interpreting how the human body guides neurite growth during development and response to injury. Further, it is of great clinical importance to identify diffusible chemical cues that promote neurite regeneration for nervous tissue repair. Despite the fast development of various types of concentration gradient generators, it has been challenging to fabricate neuron-friendly (i.e. shear-free and biocompatible for neuron growth and maturation) devices to create stable gradients, particularly for fast diffusing small molecules, which typically require high flow and shear rates. Here we present a finite element analysis for a polydimethylsiloxane/polyethylene glycol diacrylate (PDMS/PEG-DA) based gradient generator, describe the microfabrication process, and validate its use for neuronal axon polarization studies. This device provides a totally shear-free, biocompatible microenvironment with a linear and stable concentration gradient of small molecules such as forskolin. The gradient profile in this device can be customized by changing the composition or width of the PEG-DA barriers during direct UV photo-patterning within a permanently bonded PDMS device. Primary rat cortical neurons (embryonic E18) exposed to soluble forskolin gradients for 72 h exhibited statistically significant polarization and guidance of their axons. This device provides a useful platform for both chemotaxis and directional guidance studies, particularly for shear sensitive and non-adhesive cell cultures, while allowing fast new device design prototyping at a low cost.

Tuning the adhesive geometry of neurons: length and polarity control

Neurons acquire their functional and morphological axo-dendritic polarity by extending, from competing minor processes (neurites), one long axon among numerous dendrites. We employed complementary sets of micropatterns built from 2 and 6 μm wide stripes of various lengths to constrain hippocampal neuron shapes. Using these geometries, we have (i) limited the number of neuronal extensions to obtain a minimal in vitro system of bipolar neurons and (ii) controlled the neurite width during growth by the generation of a progressive cell shape asymmetry on either side of the cellular body. From this geometrical approach, we gained a high level of control of each neurite length and of the localization of axonal specification. To analyze these results, we developed a model based on a width and polarization dependent neurite elongation rate and on the existence of a critical neurite length that sets the axonal fate. Our data on the four series of micro-patterns developed for this study are described by a single set of growth parameters, well supported by experiments. The control of neuronal shapes by adhesive micro-patterns thereby offers a novel paradigm to follow the dynamical process of neurite lengthening and competition through the process of axonal polarization.

Hsp90 activity is necessary to acquire a proper neuronal polarization

Chaperones are critical for the folding and regulation of a wide array of cellular proteins. Heat Shock Proteins (Hsps) are the most representative group of chaperones. Hsp90 represents up to 1–2% of soluble protein. Although the Hsp90 role is being studied in neurodegenerative diseases, its role in neuronal differentiation remains mostly unknown. Since neuronal polarity mechanisms depend on local stability and degradation, we asked whether Hsp90 could be a regulator of axonal polarity and growth. Thus, we studied the role of Hsp90 activity in a well established model of cultured hippocampal neurons using an Hsp90 specific inhibitor, 17-AAG. Our present data shows that Hsp90 inhibition at different developmental stages disturbs neuronal polarity formation or axonal elongation. Hsp90 inhibition during the first 3h in culture promotes multiple axon morphology, while this inhibition after 3h slows down axonal elongation. Hsp90 inhibition was accompanied by decreased Akt and GSK3 expression, as well as, a reduced Akt activity. In parallel, we detected an alteration of kinesin-1 subcellular distribution. Moreover, these effects were seconded by changes in Hsp70/Hsc70 subcellular localization that seem to compensate the lack of Hsp90 activity. In conclusion, our data strongly suggests that Hsp90 activity is necessary to control the expression, activity or location of specific kinases and motor proteins during the axon specification and axon elongation processes. Even more, our data demonstrate the existence of a “time-window” for axon specification in this model of cultured neurons after which the inhibition of Hsp90 only affects axonal elongation mechanisms.

Neuropilin-1 biases dendrite polarization in the retina

The majority of neurons in the nervous system exhibit a polarized morphology, with multiple short dendrites and a single long axon. It is clear that multiple factors govern polarization in developing neurons, and the biased accumulation of intrinsic determinants to one side of the cell, coupled with responses to asymmetrically localized extrinsic factors, appears to be crucial. A number of intrinsic factors have been identified, but surprisingly little is known about the identity of the extrinsic signals. Here, we show in vivo that neuropilin-1 (Nrp1) and its co-receptor plexinA1 (Plxna1) are necessary to bias the extension of the dendrites of retinal ganglion cells to the apical side of the cell, and ectopically expressed class III semaphorins (Sema3s) disrupt this process. Importantly, the requirement for Nrp1 and Plxna1 in dendrite polarization occurs at a developmental time point after the cells have already extended their basally directed axon. Thus, we propose a novel mechanism whereby an extrinsic factor, probably a Sema3, acts through Nrp1 and Plxna1 to promote the asymmetric outgrowth of dendrites independently of axon polarization.

Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation

We have previously reported that the surface expression of K+-dependent Na+/Ca2+ exchanger 2 (NCKX2) in the somatodendritic compartment is kept low by constitutive endocytosis, which results in the polarization of surface NCKX2 to the axon. Clathrin-mediated endocytosis is initiated by interaction of the μ subunit of adaptor protein complex 2 (AP-2) with the canonical tyrosine motif (YxxΦ) of a target molecule. We examined whether endocytosis of NCKX2 involves two putative tyrosine motifs (365YGKL and 371YDTM) in the cytoplasmic loop of NCKX2. Coimmunoprecipitation assay revealed that the 365YGKL motif is essential for the interaction with the μ subunit of AP-2 (AP2M1). Consistently, either overexpression of NCKX2-Y365A mutant or knockdown of AP2M1 in cultured hippocampal neurons significantly reduced the internalization of NCKX2 from the somatodendritic surface and thus abolished the axonal polarization of surface NCKX2. Next, we tested whether the interaction between the tyrosine motif and AP2M1 is regulated by phosphorylation of the 365th tyrosine residue (Tyr-365). Tyrosine phosphorylation of heterologously expressed NCKX2-WT, but not NCKX2-Y365A, was increased by carbachol (CCh) in PC-12 cells. The effect of CCh was inhibited by PP2, a Src family kinase (SFK) inhibitor. Moreover, PP2 facilitated the endocytosis of NCKX2 in both the somatodendritic and axonal compartments, suggesting that tyrosine phosphorylation of NCKX2 by SFK negatively regulates its endocytosis. Supporting this idea, activation of SFK enhanced the NCKX activity in the proximal dendrites of dentate granule cells (GCs). These results suggest that endocytosis of somatodendritic NCKX2 is regulated by SFK-dependent phosphorylation of Tyr-365.

Centrosome positioning and primary cilia assembly orchestrate neuronal development

Establishment of axon and dendrite polarity, migration to a desired location in the developing brain, and establishment of proper synaptic connections are essential processes during neuronal development. The cellular and molecular mechanisms that govern these processes are under intensive investigation. The function of the centrosome in neuronal development has been examined and discussed in few recent studies that underscore the fundamental role of the centrosome in brain development. Clusters of emerging studies have shown that centrosome positioning tightly regulates neuronal development, leading to the segregation of cell factors, directed neurite differentiation, neuronal migration, and synaptic integration. Furthermore, cilia, that arise from the axoneme, a modified centriole, are emerging as new regulatory modules in neuronal development in conjunction with the centrosome. In this review, we focus on summarizing and discussing recent studies on centrosome positioning during neuronal development and also highlight recent findings on the role of cilia in brain development. We further discuss shared molecular signaling pathways that might regulate both centrosome and cilia associated signaling in neuronal development. Furthermore, molecular determinants such as DISC1 and LKB1 have been recently demonstrated to be crucial regulators of various aspects of neuronal development. Strikingly, these determinants might exert their function, at least in part, via the regulation of centrosome and cilia associated signaling and serve as a link between these two signaling centers. We thus include an overview of these molecular determinants.

How Morphological Constraints Affect Axonal Polarity in Mouse Neurons

Neuronal differentiation is under the tight control of both biochemical and physical information arising from neighboring cells and micro-environment. Here we wished to assay how external geometrical constraints applied to the cell body and/or the neurites of hippocampal neurons may modulate axonal polarization in vitro. Through the use of a panel of non-specific poly-L-lysine micropatterns, we manipulated the neuronal shape. By applying geometrical constraints on the cell body we provided evidence that centrosome location was not predictive of axonal polarization but rather follows axonal fate. When the geometrical constraints were applied to the neurites trajectories we demonstrated that axonal specification was inhibited by curved lines. Altogether these results indicated that intrinsic mechanical tensions occur during neuritic growth and that maximal tension was developed by the axon and expressed on straight trajectories. The strong inhibitory effect of curved lines on axon specification was further demonstrated by their ability to prevent formation of multiple axons normally induced by cytochalasin or taxol treatments. Finally we provided evidence that microtubules were involved in the tension-mediated axonal polarization, acting as curvature sensors during neuronal differentiation. Thus, biomechanics coupled to physical constraints might be the first level of regulation during neuronal development, primary to biochemical and guidance regulations.

KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2

We have previously shown that K(+)-dependent Na(+)/Ca(2+) exchanger (NCKX) is a major calcium clearance mechanism at the large axon terminals of central neurons, whereas their somata display little NCKX activity. We investigated mechanisms underlying the axonal polarization of NCKX2 in rat hippocampal neurons. We identified NCKX2 as the first neuron-specific cargo molecule of kinesin family member 21A (KIF21A). The intracellular loop of NCKX2 specifically interacted with the WD-40 repeats, a putative cargo-binding domain, of KIF21A. Dominant-negative mutant or depletion of KIF21A inhibited the transport of NCKX2-GFP to axon fibers. Knockdown of KIF21A caused calcium dysregulation at axonal boutons but not at somatodendritic regions. Despite the axonal polarization of the NCKX activity, both somatodendritic and axonal regions were immunoreactive to NCKX2. The surface expression of NCKX2 revealed by live-cell immunocytochemistry, however, displayed highly polarized distribution to the axon. Inhibition of endocytosis increased the somatodendritic surface NCKX2 and thus abolished the axonal polarization of surface NCKX2. These results indicate that KIF21A-mediated axonal transport and selective somatodendritic endocytosis underlie the axonal polarized surface expression of NCKX2.

Alternative Splicing Regulates Kv3.1 Polarized Targeting to Adjust Maximal Spiking Frequency

Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels.

Theoretical modeling for axon guidance and polarization

Theoretical modeling for axon guidance and polarization

Shaping neurons: how morphological constraints affect axonal polarity

AbstractNeuronal differentiation is under the tight control of biochemical and physical information arising from micro-environment. Here, through a panel of poly-L-lysine micropatterns, we wished to assay how external geometrical constraints of neurons may modulate axonal polarization. Constraints applied to either the cell body or to the neurite directions revealed the existence of a differential mechanical tension between the nascent axon and other neurites. Also, we show that centrosome location is not predictive of axonal polarization but responds to the force exerted by the nascent axon. Using curved trajectories for neurite growth inhibited axonal differentiation and prevented formation of multiple axons normally induced by cytochalasin or taxol treatments. Finally we provide evidence that microtubules act as curvature sensors during neuronal differentiation. Thus, biomechanics coupled to physical constraints might be the first level of regulation during neuronal development, primary to biochemical and guidance regulations.

Endocytosis and Endosomes at the Crossroads of Regulating Trafficking of Axon Outgrowth‐Modifying Receptors

In neurons, many receptors must be localized correctly to axons or dendrites for proper function. During development, receptors for nerve growth and guidance are targeted to axons and localized to growth cones where receptor activation by ligands results in promotion or inhibition of axon growth. Signaling outcomes downstream of ligand binding are determined by the location, levels and residence times of receptors on the neuronal plasma membrane. Therefore, the mechanisms controlling the trafficking of these receptors are crucial to the proper wiring of circuits. Membrane proteins accumulate on the axonal surface by multiple routes, including polarized sorting in the trans Golgi network, sorting in endosomes and removal by endocytosis. Endosomes also play important roles in the signaling pathways for both growth-promoting and -inhibiting molecules: signaling endosomes derived from endocytosis are important for signaling from growth cones to cell bodies. Growth-promoting neurotrophins and growth-inhibiting Nogo-A can use EHD4/Pincher-dependent endocytosis at the growth cone for their respective retrograde signaling. In addition to retrograde transport of endosomes, anterograde transport to axons in endosomes also occurs for several receptors, including the axon outgrowth-promoting cell adhesion molecule L1/NgCAM and TrkA. L1/NgCAM also depends on EHD4/Pincher-dependent endocytosis for its axonal polarization. In this review, we will focus on receptors whose trafficking has been reported to be modulated by the EHD4/Pincher family of endosomal regulators, namely L1/NgCAM, Trk and Nogo-A. We will first summarize the pathways underlying the axonal transport of these proteins and then discuss the potential roles of EHD4/Pincher in mediating their endocytosis.

Energy Requirements for Neuronal Polarization

Neuronal polarization lies at the heart of neuronal development, synaptic wiring, and interneuronal communication. Although much progress has been made in understanding axon growth and path finding, the mechanisms that regulate axonal neurite selection and polarity initiation remain poorly