Advances in defining signaling networks for the establishment of neuronal polarity

Neurons are highly polarized cells that have structurally and functionally distinct processes called axons and dendrites. How neurons establish polarity is one of the fundamental questions of neuroscience. In the last decade, significant progress has been made in identifying and understanding the molecular mechanisms responsible for neuronal polarization, primarily through researches conducted on cultured neurons. Advances in phosphoproteomics technologies and molecular tools have enabled comprehensive signal analysis and visualization and manipulation of signaling molecules for analyzing neuronal polarity. Furthermore, advances in gene transfer techniques have revealed the role of extracellular and intracellular signaling molecules in neuronal polarization in vivo. This review discusses the latest insights and techniques for the elucidation of the molecular mechanisms that control neuronal polarity.     

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons^1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser⁵. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response^1,3,7.We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.     

Laminin/β1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization

Axon specification during neuronal polarization is closely associated with increased microtubule stabilization in one of the neurites of unpolarized neuron, but how this increased microtubule stability is achieved is unclear. Here, we show that extracellular matrix (ECM) component laminin promotes neuronal polarization via regulating directional microtubule assembly through β1 integrin (Itgb1). Contact with laminin coated on culture substrate or polystyrene beads was sufficient for axon specification of undifferentiated neurites in cultured hippocampal neurons and cortical slices. Active Itgb1 was found to be concentrated in laminin-contacting neurites. Axon formation was promoted and abolished by enhancing and attenuating Itgb1 signaling, respectively. Interestingly, laminin contact promoted plus-end microtubule assembly in a manner that required Itgb1. Moreover, stabilizing microtubules partially prevented polarization defects caused by Itgb1 downregulation. Finally, genetic ablation of Itgb1 in dorsal telencephalic progenitors caused deficits in axon development of cortical pyramidal neurons. Thus, laminin/Itgb1 signaling plays an instructive role in axon initiation and growth, both in vitro and in vivo, through the regulation of microtubule assembly. This study has established a linkage between an extrinsic factor and intrinsic cytoskeletal dynamics during neuronal polarization.     

DIFFERENTIATION OF NEURONAL TYPES AND SYNAPSES IN MYELINATING CULTURES OF MOUSE CEREBELLUM

The Holmes silver impregnation method has made possible the recognition of multiple neuronal types and synapses in myelinating cultures of mouse cerebellum. Well stained large and medium-sized neurons are always found in small numbers near ependymal formations and are considered to be roof nuclear neurons. Neurons with poorly stained somas, abruptly demarked from intensely stained axons, are numerous and often are arranged in palisades. With prolonged maintenance in vitro these neurons develop some but not all of the features of mature Purkinje cells. A few small, densely stained, bipolar neurons, often with one process bifurcated, are found in dense regions of some cultures of newborn cerebellum. These neurons are commoner in cultures from cerebella of older mice. They closely resemble the immature granule cell in vivo. All the neuron types recognized in cultures are present in the initial explants; neurons differentiate further in vitro, but new neurons probably do not form. Synaptic boutons are found on somas and dendrites of many Purkinje cells. Two cultures contained structures resembling the basket endings which surround Purkinje cell somas in vivo. The complexity of neuronal relationships in cultures of central nervous tissue is emphasized.     

Protective effect of leptin-mediated caveolin-1 expression on neurons after spinal cord injury

Spinal cord injury (SCI) causes long-term disability and has no effective clinical treatment. After SCI, extracellular adenosine triphosphate (ATP) leads to an influx of extracellular Ca²⁺, and this Ca²⁺ overload causes neuronal toxicosis and apoptosis. The biological functions of leptin have been widely investigated in the central nervous system. In this study, we discovered that the administration of leptin could improve locomotor recovery following SCI. The aim of this study was to determine the neuroprotective mechanism of leptin in vivo and in vitro. The neuronal apoptosis and Ca²⁺ imaging signal induced by ATP were suppressed by leptin, due to elevated caveolin-1 expression. In vivo two-photon observations revealed that leptin reduced the neuronal Ca²⁺ imaging signal in the exposed spinal cords of live Thy1-YFP mice. In conclusion, leptin promotes locomotor functional recovery and suppresses neuronal impairment after SCI, suggesting that leptin has a promising clinical therapeutic value for treatment of SCI.     

Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells

BackgroundUbiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo.Conclusions/SignificanceThese data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.     

Activin A induces neuronal differentiation and survival via ALK4 in a SMAD-independent manner in a subpopulation of human neuroblastomas

Activin A is a multifunctional homo-dimeric protein that belongs to the transforming growth factor (TGF)-β superfamily. In neurons, activin has neuroprotective effects both in vitro and in vivo, but it inhibits neuronal differentiation in some cell lines. Here we report that activin A can promote neuronal differentiation in particular cases. We examined activin A-induced neuronal differentiation and survival in a selected subpopulation of a human neuroblastoma cell line, SK-N-SH, grown in low-serum (differentiation-inducing) conditions. Activin A caused dramatic neurite outgrowth, and increased the expression of neuronal markers and the transactivation of dopamine β-hydroxylase. We demonstrated that the activin A signal is transduced through the activin A type 1 receptor, ALK4, and transactivates several TGF-β target genes in a SMAD-independent manner. That is, activin A did not induce the phosphorylation of SMAD2/3, the interaction of SMAD2/3 with SMAD4, the binding of SMAD2/3 to the promoter of TGF-β target genes, or the accumulation of SMAD2/3 in the nucleus. These results suggest that, in particular cases, activin A can induce neuronal differentiation and support neuronal survival in vitro. These findings may reflect previously unknown functions of activin A in neuronal cells in vivo.     

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.     

Nanowire-Based Electrode for Acute In Vivo Neural Recordings in the Brain

We present an electrode, based on structurally controlled nanowires, as a first step towards developing a useful nanostructured device for neurophysiological measurements in vivo. The sensing part of the electrode is made of a metal film deposited on top of an array of epitaxially grown gallium phosphide nanowires. We achieved the first functional testing of the nanowire-based electrode by performing acute in vivo recordings in the rat cerebral cortex and withstanding multiple brain implantations. Due to the controllable geometry of the nanowires, this type of electrode can be used as a model system for further analysis of the functional properties of nanostructured neuronal interfaces in vivo.     

Early Increase and Late Decrease of Purkinje Cell Dendritic Spine Density in Prion-Infected Organotypic Mouse Cerebellar Cultures

Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4–5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.     

Microglia activation-induced mesencephalic dopaminergic neurodegeneration — an in vitro model for Parkinson’s disease

Uncontrolled and chronic microglia activation has been implicated in the process of dopaminergic neuron degeneration in sporadic Parkinson??s disease (PD). Elevated proinflammatory mediators, presumably from activated microglia (e.g., cytokines, PGE₂, nitric oxide, and superoxide radical), have been observed in PD patients and are accompanied by dopaminergic neuronal loss. Preclinical studies have demonstrated the deleterious effects of proinflammatory mediators in various in vivo and in vitro models of PD. The use of in vitro studies provides a unique tool to investigate the interaction between neurons and microglia and is especially valuable when considering the role of activated microglia in neuronal death. Here we summarize findings highlighting the potential mechanisms of microgliamediated neurodegeneration in PD.     

Drosophila Hrp48 Is Required for Mushroom Body Axon Growth,Branching and Guidance

RNA binding proteins assemble on mRNAs to control every single step of their life cycle, from nuclear splicing to cytoplasmic localization, stabilization or translation. Consistent with an essential role of RNA binding proteins in neuronal maturation and function, mutations in this class of proteins, in particular in members of the hnRNP family, have been associated with neurological diseases. To date, however, the physiological function of hnRNPs during in vivo neuronal development has remained poorly explored. Here, we have investigated the role of Drosophila Hrp48, a fly homologue of mammalian hnRNP A2/B1, during central nervous system development. Using a combination of mutant conditions, we showed that hrp48 is required for the formation, growth and guidance of axonal branches in Mushroom Body neurons. Furthermore, our results revealed that hrp48 inactivation induces an overextension of Mushroom Body dorsal axonal branches, with a significantly higher penetrance in females than in males. Finally, as demonstrated by immunolocalization studies, Hrp48 is confined to Mushroom Body neuron cell bodies, where it accumulates in the cytoplasm from larval stages to adulthood. Altogether, our data provide evidence for a crucial in vivo role of the hnRNP Hrp48 in multiple aspects of axon guidance and branching during nervous system development. They also indicate cryptic sex differences in the development of sexually non-dimorphic neuronal structures.     

Differentiation of human ES cell-derived neural progenitors to neuronal cells with regional specific identity by co-culturing of notochord and somite

Limitations to the in vivo study of human nervous system development make it necessary to design an in vitro model to evaluate the in vivo effects of surrounding tissues on neurogenesis and regional identity of the human neural plate. Rostral neural progenitors (NPs) were initially generated from adherent human embryonic stem cells (hESCs) in a defined condition and characterized. Then, to find the role of somites (S) and notochords (N) in rostro-caudal (RC) and dorso-ventral (DV) patterning of neuronal cells, NPs were co-cultured with microencapsulated chicken S or N in alginate beads. In this study, N induced more neurogenesis as evaluated by expression of TUJ1 and MAP2-positive cells. Additionally, N induced spinal cord ventral brachiothoracic identity as well as NPs proliferation. We observed a synergic effect on motoneuron induction when S and N were used together. Moreover, S induced hindbrain identity in differentiated neuronal cells from NPs. These results indicate that highly enriched NPs can be generated in an adherent and defined system from hESCs. Moreover, S and N tissues highly influenced neuronal differentiation in point of proliferation, neurogenesis, and RC and DV regional identity. These results indicate a very simple and efficient protocol to mimic in vivo events of human neural development in vitro which is important in the context of developmental neuroscience and cellular replacement therapies.     

Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors

The cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM⁺ enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40 days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM⁺ enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells.     

Inhibition of Bax protects neuronal cells from oligomeric A�� neurotoxicity

Although oligomeric β-amyloid (Aβ) has been suggested to have an important role in Alzheimer disease (AD), the mechanism(s) of how Aβ induces neuronal cell death has not been fully identified. The balance of pro- and anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2 and Bcl-w versus Bad, Bim and Bax) has been known to have a role in neuronal cell death and, importantly, expression levels of these proteins are reportedly altered in the vulnerable neurons in AD. However, the roles of apoptotic proteins in oligomeric Aβ-induced cell death remain unclear in vivo or in more physiologically relevant models. In addition, no study to date has examined whether Bax is required for the toxicity of oligomeric Aβ. Here, we found that treatment with oligomeric Aβ increased Bim levels but decreased Bcl-2 levels, leading to the activation of Bax and neuronal cell death in hippocampal slice culture and in vivo. Furthermore, the inhibition of Bax activity either by Bax-inhibiting peptide or bax gene knockout significantly prevented oligomeric Aβ-induced neuronal cell death. These findings are first to demonstrate that Bax has an essential role in oligomeric Aβ-induced neuronal cell death, and that the targeting of Bax may be a therapeutic approach for AD.     

Clonal Human Fetal Ventral Mesencephalic Dopaminergic Neuron Precursors for Cell Therapy Research

A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH⁺ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experiments.     

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals^1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo^3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex^5-9.This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.     

RNA Binding Protein Rbms1 Enables Neuronal Differentiation and Radial Migration during Neocortical Development by Binding and Stabilizing the RNA Message for Efr3a

Various RNA-binding proteins (RBPs) are key components in RNA metabolism and contribute to several neurodevelopmental disorders. To date, only a few of such RBPs have been characterized for their roles in neocortex development. Here, we show that the RBP, Rbms1, is required for radial migration, polarization and differentiation of neuronal progenitors to neurons in the neocortex development. Rbms1 expression is highest in the early development in the developing cortex, with its expression gradually diminishing from embryonic day 13.5 (E13.5) to postnatal day 0 (P0). From in utero electroporation (IUE) experiments when Rbms1 levels are knocked down in neuronal progenitors, their transition from multipolar to bipolar state is delayed and this is accompanied by a delay in radial migration of these cells. Reduced Rbms1 levels in vivo also reduces differentiation as evidenced by a decrease in levels of several differentiation markers, meanwhile having no significant effects on proliferation and cell cycle rates of these cells. As an RNA binding protein, we profiled the RNA binders of Rbms1 by a cross-linked-RIP sequencing assay, followed by quantitative real-time polymerase chain reaction verification and showed that Rbms1 binds and stabilizes the mRNA for Efr3a, a signaling adapter protein. We also demonstrate that ectopic Efr3a can recover the cells from the migration defects due to loss of Rbms1, both in vivo and in vitro migration assays with cultured cells. These imply that one of the functions of Rbms1 involves the stabilization of Efr3a RNA message, required for migration and maturation of neuronal progenitors in radial migration in the developing neocortex.     

Substrate Topography Determines Neuronal Polarization and Growth In Vitro

The establishment of neuronal connectivity depends on the correct initial polarization of the young neurons. In vivo, developing neurons sense a multitude of inputs and a great number of molecules are described that affect their outgrowth. In vitro, many studies have shown the possibility to influence neuronal morphology and growth by biophysical, i.e. topographic, signaling. In this work we have taken this approach one step further and investigated the impact of substrate topography in the very early differentiation stages of developing neurons, i.e. when the cell is still at the round stage and when the first neurite is forming. For this purpose we fabricated micron sized pillar structures with highly reproducible feature sizes, and analyzed neurons on the interface of flat and topographic surfaces. We found that topographic signaling was able to attract the polarization markers of mouse embryonic neurons -N-cadherin, Golgi-centrosome complex and the first bud were oriented towards topographic stimuli. Consecutively, the axon was also preferentially extending along the pillars. These events seemed to occur regardless of pillar dimensions in the range we examined. However, we found differences in neurite length that depended on pillar dimensions. This study is one of the first to describe in detail the very early response of hippocampal neurons to topographic stimuli.     

Motor domain-mediated autoinhibition dictates axonal transport by the kinesin UNC-104/KIF1A

The UNC-104/KIF1A motor is crucial for axonal transport of synaptic vesicles, but how the UNC-104/KIF1A motor is activated in vivo is not fully understood. Here, we identified point mutations located in the motor domain or the inhibitory CC1 domain, which resulted in gain-of-function alleles of unc-104 that exhibit hyperactive axonal transport and abnormal accumulation of synaptic vesicles. In contrast to the cell body localization of wild type motor, the mutant motors accumulate on neuronal processes. Once on the neuronal process, the mutant motors display dynamic movement similarly to wild type motors. The gain-of-function mutation on the motor domain leads to an active dimeric conformation, releasing the inhibitory CC1 region from the motor domain. Genetically engineered mutations in the motor domain or CC1 of UNC-104, which disrupt the autoinhibitory interface, also led to the gain of function and hyperactivation of axonal transport. Thus, the CC1/motor domain-mediated autoinhibition is crucial for UNC-104/KIF1A-mediated axonal transport in vivo.