GPI‐anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro

The glycosyl phosphatidylinositol (GPI) lipid anchor, which directs GPI‐anchored proteins to the apical cell surface in certain polarized epithelial cell types, has been proposed to act as an axonal protein targeting signal in neurons. However, as several GPI‐anchored proteins have been found on both the axonal and somatodendritic cell‐surface domains of a variety of neuronal cell types, the role of the GPI anchor in protein localization to the axon remains unclear. To begin to address the role of the GPI anchor in neuronal protein localization, we used a replication‐incompetent retroviral vector to express a model GPI‐anchored protein, human placental alkaline phosphatase (hPLAP), in early postnatal mouse cerebellar granule neurons developing in vitro. Purified granule neurons were cultured in large mitotically active cellular reaggregates to allow retroviral infection of undifferentiated, proliferating granule neuron precursors. To more easily visualize hPLAP localization during the sequence of differentiation of single postmitotic granule neurons, reaggregates were dissociated following infection, plated as high‐density monolayers, and maintained for 1–9 days under serum‐free culture conditions. As we previously demonstrated for uninfected granule neurons developing in monolayer culture, hPLAP‐expressing granule neurons likewise developed in vitro through a series of discrete temporal stages highly similar to those observed in situ. hPLAP‐expressing granule neurons first extended either a single neurite or two axonal processes, and subsequently attained a mature, well‐polarized morphology consisting of multiple short dendrites and one or two axons that extended up to 3 mm across the culture substratum. hPLAP was expressed uniformly on the entire cell surface at each stage of granule neuron differentiation. Thus, it appears that the GPI anchor is not sufficient to confer axonal localization to an exogenous GPI‐anchored protein expressed in a well‐polarized primary neuronal cell type in vitro; other signals, such asthose present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon‐specific GPI‐anchored proteins. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 119–141, 1999     

AMP‐activated protein kinase mediates activity‐dependent axon branching by recruiting mitochondria to axon

During development, axons are guided to their target areas and provide local branching. Spatiotemporal regulation of axon branching is crucial for the establishment of functional connections between appropriate pre‐ and postsynaptic neurons. Common understanding has been that neuronal activity contributes to the proper axon branching; however, intracellular mechanisms that underlie activity‐dependent axon branching remain elusive. Here, we show, using primary cultures of the dentate granule cells, that neuronal depolarization‐induced rebalance of mitochondrial motility between anterograde versus retrograde transport underlies the proper formation of axonal branches. We found that the depolarization‐induced branch formation was blocked by the uncoupler p‐trifluoromethoxyphenylhydrazone, which suggests that mitochondria‐derived ATP mediates the observed phenomena. Real‐time analysis of mitochondrial movement defined the molecular mechanisms by showing that the pharmacological activation of AMP‐activated protein kinase (AMPK) after depolarization increased anterograde transport of mitochondria into axons. Simultaneous imaging of axonal morphology and mitochondrial distribution revealed that mitochondrial localization preceded the emergence of axonal branches. Moreover, the higher probability of mitochondrial localization was correlated with the longer lifetime of axon branches. We qualitatively confirmed that neuronal ATP levels decreased immediately after depolarization and found that the phosphorylated form of AMPK was increased. Thus, this study identifies a novel role for AMPK in the transport of axonal mitochondria that underlie the neuronal activity‐dependent formation of axon branches. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 557–573, 2014     

Neurobiology of the basal platyhelminth <Emphasis Type="Italic">Macrostomum lignano</Emphasis>: map and digital 3D model of the juvenile brain neuropile

We have analyzed brain structure in Macrostomum lignano, a representative of the basal platyhelminth taxon Macrostomida. Using confocal microscopy and digital 3D modeling software on specimens labeled with general markers for neurons (tyrTub), muscles (phalloidin), and nuclei (Sytox), an atlas and digital model of the juvenile Macrostomum brain was generated. The brain forms a ganglion with a central neuropile surrounded by a cortex of neuronal cell bodies. The neuropile contains a stereotypical array of compact axon bundles, as well as branched terminal axons and dendrites. Muscle fibers penetrate the flatworm brain horizontally and vertically at invariant positions. Beside the invariant pattern of neurite bundles, these “cerebral muscles” represent a convenient system of landmarks that help define discrete compartments in the juvenile brain. Commissural axon bundles define a dorsal and ventro-medial neuropile compartment, respectively. Longitudinal axons that enter the neuropile through an invariant set of anterior and posterior nerve roots define a ventro-basal and a central medial compartment in the neuropile. Flanking these “fibrous” compartments are neuropile domains that lack thick axon bundles and are composed of short collaterals and terminal arborizations of neurites. Two populations of neurons, visualized by antibodies against FMRFamide and serotonin, respectively, were mapped relative to compartment boundaries. This study will aid in the documentation and interpretation of patterns of gene expression, as well as functional studies, in the developing Macrostomum brain.     

Distinct roles for Robo2 in the regulation of axon and dendrite growth by retinal ganglion cells

Guidance factors act on the tip of a growing axon to direct it to its target. What role these molecules play, however, in the control of the dendrites that extend from that axon’s cell body is poorly understood. Slits, through their Robo receptors, guide many types of axons, including those of retinal ganglion cells (RGCs). Here we assess and contrast the role of Slit/Robo signalling in the growth and guidance of the axon and dendrites extended by RGCs in Xenopus laevis. As Xenopus RGCs extend dendrites, they express robo2 and robo3, while slit1 and slit2 are expressed in RGCs and in the adjacent inner nuclear layer. Interestingly, our functional data with antisense knockdown and dominant negative forms of Robo2 (dnRobo2) and Robo3 (dnRobo3) indicate that Slit/Robo signalling has no role in RGC dendrite guidance, and instead is necessary to stimulate dendrite branching, primarily via Robo2. Our in vitro culture data argue that Slits are the ligands involved. In contrast, both dnRobo2 and dnRobo3 inhibited the extension of axons and caused the misrouting of some axons. Based on these data, we propose that Robo signalling can have distinct functions in the axon and dendrites of the same cell, and that the specific combinations of Robo receptors could underlie these differences. Slit acts via Robo2 in dendrites as a branching/growth factor but not in guidance, while Robo2 and Robo3 function in concert in axons to mediate axonal interactions and respond to Slits as guidance factors. These data underscore the likelihood that a limited number of extrinsic factors regulate the distinct morphologies of axons and dendrites.     

Identification of an axonal determinant in the C-terminus of the sodium channel Na(v)1.2

To obtain a better understanding of how hippocampal neurons selectively target proteins to axons, we assessed whether any of the large cytoplasmic regions of neuronal sodium channel Na(v)1.2 contain sufficient information for axonal compartmentalization. We show that addition of the cytoplasmic C-terminal region of Na(v)1.2 restricted the distribution of a dendritic-axonal reporter protein to axons. The analysis of mutants revealed that a critical segment of nine amino acids encompassing a di-leucine-based motif mediates axonal compartmentalization of chimera. In addition, the Na(v)1.2 C-terminus is recognized by the clathrin endocytic pathway both in non-neuronal cells and the somatodendritic domain of hippocampal neurons. The mutation of the di-leucine motif located within the nine amino acid sequence to alanines resulted in the loss of chimera compartmentalization in axons and of internalization. These data suggest that selective elimination by endocytosis in dendrites may account for the compartmentalized distribution of some proteins in axons.     

Axon guidance and neuronal migration research in China

Proper migration of neuronal somas and axonal growth cones to designated locations in the developing brain is essential for the assembly of functional neuronal circuits.Rapid progress in research of axon guidance and neuronal migration has been made in the last twenty years.Chinese researchers began their exploration in this field ten years ago and have made significant contributions in clarifying the signal transduction of axon guidance and neuronal migration.Several unique experimental approaches,includin...     

Development of polarity in cerebellar granule neurons

Axon formation in developing cerebellar granule neurons in situ is spatially and temporally segregated from subsequent neuronal migration and dendrite formation. To examine the role of local environmental cues on early steps in granule cell differentiation, the sequence of morphologic development and polarized distribution of membrane proteins was determined in granule cells isolated from contact with other cerebellar cell types. Granule cells cultured at low density developed their characteristic axonal and dendritic morphologies in a series of discrete temporal steps highly similar to those observed in situ, first extending a unipolar process, then long, thin bipolar axons, and finally becoming multipolar, forming short dendrites around the cell body. Axonal- and dendritic-specific cytoskeletal markers were segregated to the morphologically distinct domains. The cell surface distribution of a specific class of endogenous glycoproteins, those linked to the membrane by a glycosylphosphatidyl inositol (GPI) anchor, was also examined. The GPI-anchored protein, TAG-1, which is segregated to the parallel fiber axons in situ, was found exclusively on granule cell axons in vitro; however, two other endogenous GPI-anchored proteins were found on both the axonal and somatodendritic domains. These results demonstrate that granule cells develop polarity in a cell type-specific manner in the absence of the spatial cues of the developing cerebellar cortex. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 223–236, 1997.     

Synaptotagmin‐1 promotes the formation of axonal filopodia and branches along the developing axons of forebrain neurons

Synaptotagmin‐1 (syt1) is a Ca²⁺‐binding protein that functions in regulation of synaptic vesicle exocytosis at the synapse. Syt1 is expressed in many types of neurons well before synaptogenesis begins both in vivo and in vitro. To determine if expression of syt1 has a functional role in neuronal development before synapse formation, we examined the effects of syt1 overexpression and knockdown on the growth and branching of the axons of cultured primary embryonic day 8 chicken forebrain neurons. In vivo these neurons express syt1, and most have not yet extended axons. We present evidence that syt1 plays a role in regulating axon branching, while not regulating overall axon length. To study the effects of overexpression of syt1, we used adenovirus‐mediated infection to introduce a syt1‐YFP construct, or control GFP construct, into neurons. Syt1 levels were reduced using RNA interference. Overexpression of syt1 increased the formation of axonal filopodia and branches. Conversely, knockdown of syt1 decreased the number of axonal filopodia and branches. Time‐lapse analysis of filopodial dynamics in syt1‐overexpressing cells demonstrated that elevation of syt1 levels increased both the frequency of filopodial initiation and their lifespan. Taken together these data indicate that syt1 regulates the formation of axonal filopodia and branches before engaging in its conventional functions at the synapse. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity

The development of a polarised morphology with multiple dendrites and a single axon is an essential step in the differentiation of neurons. The establishment of neuronal polarity is directed by the sequential activity of the GTPases Rap1B and Cdc42. Rap1B is initially present in all neurites of unpolarised neurons, but becomes restricted to the tip of a single process during the establishment of neuronal polarity where it specifies axonal identity. Here, we show that the ubiquitin ligases Smad ubiquitination regulatory factor-1 (Smurf1) and Smurf2 are essential for neurite growth and neuronal polarity, respectively, and regulate the GTPases Rho and Rap1B in hippocampal neurons. Smurf2 is required for the restriction of Rap1B to a single neurite. Smurf2 ubiquitinates inactive Rap1B and initiates its degradation through the ubiquitin/proteasome pathway (UPS). Degradation of Rap1B restricts it to a single neurite and thereby ensures that neurons extend a single axon.     

Plasticity of polarization: changing dendrites into axons in neurons integrated in neuronal circuits

Developing neurons can change axonal and dendritic fate upon axonal lesion, but it is unclear whether neurons retain such plasticity when they are synaptically interconnected. To address whether polarity is reversible in mature neurons, we cut the axon of GFP-labeled hippocampal neurons in dissociated and organotypic cultures and found that a new axon arose from a mature dendrite. The regenerative response correlated with the length of the remaining stump: proximal axotomies (<35 microm) led to the transformation of a dendrite into an axon (identity change), whereas distal cuts (>35 microm) induced axon regrowth, similar to what is seen in young neurons. Searching for a putative landmark in the distal axon that could determine axon identity, we focused on the stability of microtubules, which regulate initial neuronal polarization during early development. We found that functionally polarized neurons contain a distinctively high proportion of stable microtubules in the distal axon. Moreover, pharmacological stabilization of microtubules was sufficient to induce the formation of multiple axons out of differentiated dendrites. Our data argue that mature neurons integrated in functional networks remain flexible in their polarity and that mechanisms acting during initial axon selection can be reactivated to induce axon growth out of functionally mature dendrites.     

Neuronal Polarity

The assembly of functional neuronal networks in the developing animal relies on the polarization of neurons, i.e., the formation of a single axon and multiple dendrites. Breaking the symmetry of neurons depends on cytoskeletal rearrangements. In particular, axon specification requires local dynamic instability of actin and stabilization of microtubules. The polarized cytoskeleton also provides the basis for selective trafficking and retention of cellular components in the future somatodendritic or axonal compartments. Hence, these mechanisms are not only essential to achieve neuronal polarization, but also to maintain it. Different extracellular and intracellular signals converge on the regulation of the cytoskeleton. Most notably, Rho GTPases, PI3K, Ena/VASP, cofilin and SAD kinases are major intracellular regulators of neuronal polarity. Analyzing polarity signals under physiological conditions will provide a better understanding of how neurons can be induced to repolarize under pathological conditions, i.e., to regenerate their axons after central nervous system (CNS) injury.One ambitious aim in cellular biology is to unravel the molecular mechanisms driving cellular asymmetry and polarization. The polarity of neurons is particularly dramatic as neurons undergo complex morphological rearrangements to assemble into neuronal circuits and propagate signals. They start as round neuronal spheres, gradually adopting a complex morphology by forming one long axon and several shorter dendrites to eventually connect to other neurons via synapses. Neuronal compartments segregate into molecularly and functionally distinct zones. For example, signal input takes place at the postsynaptic densities where a chemical signal elicits electric postsynaptic potentials. These potentials are integrated along the dendritic tree and cell body to trigger an action potential arising at the axon hillock and propagating further along the axon. At their terminals, the electrical signal is reconverted into a chemical signal by the release of synaptic vesicles containing neurotransmitter.Neurons maintain their polarity throughout life by different intracellular mechanisms and molecular signals. During the last decade, cell biological and molecular approaches helped to discover many of the molecules and signaling mechanisms regulating neuronal polarity (Yoshimura et al. 2006; Arimura and Kaibuchi 2007; Witte and Bradke 2008). The aim of this article is to summarize the current knowledge and principles of breaking neuronal symmetry to generate functional neurons, and to discuss the future challenges in the field. The article covers two different topics: intrinsic mechanisms that govern symmetry breaking in the absence of external cues (in vitro systems) and the role of extracellular signaling in the establishment of neuronal polarity in vivo.     

Neuronal ribonucleoprotein granules: Dynamic sensors of localized signals

Membrane‐less organelles, because of their capacity to dynamically, selectively and reversibly concentrate molecules, are very well adapted for local information processing and rapid response to environmental fluctuations. These features are particularly important in the context of neuronal cells, where synapse‐specific activation, or localized extracellular cues, induce signaling events restricted to specialized axonal or dendritic subcompartments. Neuronal ribonucleoprotein (RNP) particles, or granules, are nonmembrane bound macromolecular condensates that concentrate specific sets of mRNAs and regulatory proteins, promoting their long‐distance transport to axons or dendrites. Neuronal RNP granules also have a dual function in regulating the translation of associated mRNAs: while preventing mRNA translation at rest, they fuel local protein synthesis upon activation. As revealed by recent work, rapid and reversible switches between these two functional modes are triggered by modifications of the networks of interactions underlying RNP granule assembly. Such flexible properties also come with a cost, as neuronal RNP granules are prone to transition into pathological aggregates in response to mutations, aging, or cellular stresses, further emphasizing the need to better understand the mechanistic principles governing their dynamic assembly and regulation in living systems.     

Dose and age‐dependent axonal responses of embryonic trigeminal neurons to localized NGF via p75NTR receptor

Nerve growth factor (NGF) and related neurotrophins are target‐derived survival factors for sensory neurons. In addition, these peptides modulate neuronal differentiation, axon guidance, and synaptic plasticity. We tested axonal behavior of embryonic trigeminal neurons towards localized sources of NGF in collagen gel assays. Trigeminal axons preferentially grow towards lower doses of localized NGF and grow away from higher concentrations at earlier stages of development, but do not show this response later. Dorsal root ganglion axons also show similar responses to NGF, but NGF‐dependent superior cervical ganglion axons do not. Such axonal responses to localized NGF sources were also observed in Bax^−/− mice, suggesting that the axonal effects are largely independent of cell survival. Immunocytochemical studies indicated that axons, which grow towards or away from localized NGF are TrkA‐positive, and TrkA^−/− TG axons do not respond to any dose of NGF. We further show that axonal responses to NGF are absent in TG derived from mice that lack the p75 neurotrophin receptor (p75^NTR). Collectively, our results suggest that localized sources of NGF can direct axon outgrowth from trigeminal ganglion in a dose‐ and age‐dependent fashion, mediated by p75^NTR signaling through TrkA expressing axons. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Correlative single‐molecule localization microscopy and electron tomography reveals endosome nanoscale domains

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction‐limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single‐molecule photo‐switching are opposed. Here, we developed a novel superCLEM workflow that combines triple‐color SMLM (dSTORM & PALM) and electron tomography using semi‐thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub‐compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.     

Neuronal Compartmentalization: A Means to Integrate Sensory Input at the Earliest Stage of Information Processing?

In numerous peripheral sense organs, external stimuli are detected by primary sensory neurons compartmentalized within specialized structures composed of cuticular or epithelial tissue. Beyond reflecting developmental constraints, such compartmentalization also provides opportunities for grouped neurons to functionally interact. Here, the authors review and illustrate the prevalence of these structural units, describe characteristics of compartmentalized neurons, and consider possible interactions between these cells. This article discusses instances of neuronal crosstalk, examples of which are observed in the vertebrate tastebuds and multiple types of arthropod chemosensory hairs. Particular attention is paid to insect olfaction, which presents especially well-characterized mechanisms of functional, cross-neuronal interactions. These examples highlight the potential impact of peripheral processing, which likely contributes more to signal integration than previously considered. In surveying a wide variety of structural units, it is hoped that this article will stimulate future research that determines whether grouped neurons in other sensory systems can also communicate to impact information processing.     

Ultrastructural parameters of GABA-ergic and non-GABA-ergic synaptic contacts on neurons of the catsubstantia nigra

Nigrothalamic neurons were identified in the reticular part of thesubstantia nigra using labelling by the retrograde axonal transport of horseradish peroxidase. Nine parameters of the synaptic contacts (n=195) were analyzed, including the size and shape of terminals and size and type of synaptic vesicies. Sixty-six percent of axon terminals studied formed symmetric contacts and contained large polymorphic vesicles (group I). Two-thirds of these synapses were located on the distal dendrites, while one-third was distributed on the perikarya and proximal dendrites. Synapses of group II (29% of all synapses analyzed) exhibited asymmetric contacts and contained round agranular vesicles. Among these synapses, 79% were located on the distal dendrites, 19% were located on the proximal dendrites, and only 2% were located on the neuronal perikarya. Axon terminals of group III (5% of total population) exhibited symmetric contact and contained small polymorphic vesicles. High-resolution immunogold EM histochemistry indicated that 63% of the group-I axon terminals were GABA-positive. The majority of synapses on the labelled nigrothalamic neurons (21 contacts of 25) belonged to group I. Among these 21 synapses, 19 were axo-somatic and mostly GABA-positive. Within group II, 30% of synapses showed slightly expressed GABA-positivity.Neirofiziologiya/Neurophysiology, Vol. 27, No. 2, pp. 147–157, March–April, 1995.