Spatial confinement: A spur for axonal growth.

The concept of spatial confinement is the basis of cell positioning and guidance in in vitro studies. In vivo, it reflects many situations faced during embryonic development. In vitro, spatial confinement of neurons is achieved using different technological approaches: adhesive patterning, topographical structuring, microfluidics and the use of hydrogels. The notion of chemical or physical frontiers is particularly central to the behaviors of growth cones and neuronal processes under confinement. They encompass phenomena of cell spreading, boundary crossing, and path finding on surfaces with different adhesive properties. However, the most universal phenomenon related to confinement, regardless of how it is implemented, is the acceleration of neuronal growth. Overall, a bi-directional causal link emerges between the shape of the growth cone and neuronal elongation dynamics, both in vivo and in vitro. The sensing of adhesion discontinuities by filopodia and the subsequent spatial redistribution and size adaptation of these actin-rich filaments seem critical for the growth rate in conditions in which adhesive contacts and actin-associated clutching forces dominate. On the other hand, the involvement of microtubules, specifically demonstrated in 3D hydrogel environments and leading to ameboid-like locomotion, could be relevant in a wider range of growth situations. This review brings together a literature collected in distinct scientific fields such as development, mechanobiology and bioengineering that highlight the consequences of confinement and raise new questions at different cellular scales. Its ambition is to stimulate new research that could lead to a better understanding of what gives neurons their ability to establish and regulate their exceptional size.

Actin cytoskeletal dynamics do not impose an energy drain on growth cone bioenergetics.

The regulation of the intracellular level of ATP is a fundamental aspect of bioenergetics. Actin cytoskeletal dynamics have been reported to be an energetic drain in developing neurons and platelets. We addressed the role of actin dynamics in primary embryonic chicken neurons using luciferase assays, and by measurement of the ATP/ADP ratio using the ratiometric reporter PercevalHR and the ATP level using the ratiometric reporter mRuby-iATPSnFR. None of the methods revealed an effect of suppressing actin dynamics on the decline in the neuronal ATP level or the ATP/ADP ratio following shutdown of ATP production. Similarly, we find that treatments that elevate or suppress actin dynamics do not alter the ATP/ADP ratio in growth cones, the subcellular domain with the highest actin dynamics in developing neurons. Collectively, the data indicate that actin cytoskeletal dynamics are not a significant energy drain in developing neurons and that the ATP/ADP ratio is maintained when energy utilization varies. Discrepancies between prior work and the current data are discussed with emphasis on methodology and interpretation of the data.

Septins mediate a microtubule-actin crosstalk that enables actin growth on microtubules.

Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin. Septins are filamentous guanosine-5'-triphosphate (GTP) binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin, and intermediate filaments. Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Superresolution and platinum replica electron microscopy (PREM) show that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We demonstrate that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings show that septins directly mediate microtubule interactions with actin filaments, and reveal a mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.

Ribosomal Protein Dynamics and Its Association with Actin Filaments and Local Translation in Axonal Growth Cones of Dorsal Root Ganglia Neurons.

Local translation in growth cones plays a critical role in responses to extracellular stimuli, such as axon guidance cues. We previously showed that brain-derived neurotrophic factor activates translation and enhances novel protein synthesis through the activation of mammalian target of rapamycin complex 1 signaling in growth cones of dorsal root ganglion neurons. In this study, we focused on 40S ribosomal protein S6 (RPS6), 60S ribosomal protein P0/1/2 (RPP0/1/2), and actin filaments to determine how localization of ribosomal proteins changes with overall protein synthesis induced by neurotrophins. Our quantitative analysis using immunocytochemistry and super-resolution microscopy indicated that RPS6, RPP0/1/2, and actin tend to colocalize in the absence of stimulation, and that these ribosomal proteins tend to dissociate from actin and associate with each other when local protein synthesis is enhanced. We propose that this is because stimulation causes ribosomal subunits to associate with each other to form actively translating ribosomes (polysomes). This study further clarifies the role of cytoskeletal components in local translation in growth cones.

XMAP215 promotes microtubule-F-actin interactions to regulate growth cone microtubules during axon guidance in Xenopuslaevis.

It has long been established that neuronal growth cone navigation depends on changes in microtubule (MT) and F-actin architecture downstream of guidance cues. However, the mechanisms by which MTs and F-actin are dually coordinated remain a fundamentally unresolved question. Here, we report that the well-characterized MT polymerase, XMAP215 (also known as CKAP5), plays an important role in mediating MT-F-actin interaction within the growth cone. We demonstrate that XMAP215 regulates MT-F-actin alignment through its N-terminal TOG 1-5 domains. Additionally, we show that XMAP215 directly binds to F-actin in vitro and co-localizes with F-actin in the growth cone periphery. We also find that XMAP215 is required for regulation of growth cone morphology and response to the guidance cue, Ephrin A5. Our findings provide the first strong evidence that XMAP215 coordinates MT and F-actin interaction in vivo We suggest a model in which XMAP215 regulates MT extension along F-actin bundles into the growth cone periphery and that these interactions may be important to control cytoskeletal dynamics downstream of guidance cues. This article has an associated First Person interview with the first author of the paper.

Synapse formation: from cellular and molecular mechanisms to neurodevelopmental and neurodegenerative disorders.

The precise patterns of neuronal assembly during development determine all functional outputs of a nervous system; these may range from simple reflexes to learning, memory, cognition, etc. To understand how brain functions and how best to repair it after injury, disease, or trauma, it is imperative that we first seek to define fundamental steps mediating this neuronal assembly. To acquire the sophisticated ensemble of highly specialized networks seen in a mature brain, all proliferated and migrated neurons must extend their axonal and dendritic processes toward targets, which are often located at some distance. Upon contact with potential partners, neurons must undergo dramatic structural changes to become either a pre- or a postsynaptic neuron. This connectivity is cemented through specialized structures termed synapses. Both structurally and functionally, the newly formed synapses are, however, not static as they undergo consistent changes in order for an animal to meet its behavioral needs in a changing environment. These changes may be either in the form of new synapses or an enhancement of their synaptic efficacy, referred to as synaptic plasticity. Thus, synapse formation is not restricted to neurodevelopment; it is a process that remains active throughout life. As the brain ages, either the lack of neuronal activity or cell death render synapses dysfunctional, thus giving rise to neurodegenerative disorders. This review seeks to highlight salient steps that are involved in a neuron's journey, starting with the establishment, maturation, and consolidation of synapses; we particularly focus on identifying key players involved in the synaptogenic program. We hope that this endeavor will not only help the beginners in this field to understand how brain networks are assembled in the first place but also shed light on various neurodevelopmental, neurological, neurodegenerative, and neuropsychiatric disorders that involve synaptic inactivity or dysfunction.

Rapid Growth Cone Uptake and Dynein-Mediated Axonal Retrograde Transport of Negatively Charged Nanoparticles in Neurons Is Dependent on Size and Cell Type.

Nanoparticles (NPs) are now used in numerous technologies and serve as carriers for several new classes of therapeutics. Studies of the distribution of NPs in vivo demonstrate that they can be transported through biological barriers and are concentrated in specific tissues. Here, transport behavior, and final destination of polystyrene NPs are reported in primary mouse cortical neurons and SH-SY5Y cells, cultured in two-compartmental microfluidic devices. In both cell types, negative polystyrene NPs (PS(-)) smaller than 100 nm are taken up by the axons, undergo axonal retrograde transport, and accumulate in the somata. Examination of NP transport reveals different transport mechanisms depending on the cell type, particle charge, and particle internalization by the lysosomes. In cortical neurons, PS(-) inside lysosomes and 40 nm positive polystyrene NPs undergo slow axonal transport, whereas PS(-) outside lysosomes undergo fast axonal transport. Inhibition of dynein in cortical neurons decreases the transport velocity and cause a dose-dependent reduction in the number of accumulated PS(-), suggesting that the fast axonal transport is dynein mediated. These results show that the axonal retrograde transport of NPs depends on the endosomal pathway taken and establishes a means for screening nanoparticle-based therapeutics for diseases that involve neurons.

Plastin 3 is upregulated in iPSC-derived motoneurons from asymptomatic SMN1-deleted individuals.

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier.

Control over Neurite Directionality and Neurite Elongation on Anisotropic Micropillar Arrays.

Control over neurite orientation in primary hippocampal neurons is achieved by using interrupted, anisotropic micropillar arrays as a cell culture platform. Both neurite orientation and neurite length are controlled by a function of interpillar distance.

Using Cultured Mammalian Neurons to Study Cellular Processes and Neurodegeneration: A Suite of Undergraduate Lab Exercises.

Cell culture is a powerful tool for exploring cellular function. Culturing primary neurons has revealed how neurons communicate in learning and memory (Kandel, 2006) and provided insights into the mechanisms of neurodegenerative diseases such as Parkinson's and Alzheimer's disease (Alberio et al., 2012; Trinchese, et al., 2004). Here we describe a series of four modular laboratory exercises to integrate this neuroscience technique in undergraduate teaching laboratories. First, we describe the modular approach. Then we provide educators with simple techniques for culturing rat primary neurons, performing immunohistochemistry to label cellular components, and illustrating neurodegeneration caused by reactive oxygen species. We describe teaching exercises that culminate in student-generated research projects. Finally, we describe potential barriers students may face when integrating modern cell culture experiments into teaching laboratories.

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.

Rufy3, a protein specifically expressed in neurons, interacts with actin-bundling protein Fascin to control the growth of axons.

For our nervous system to function properly, each neuron must generate a single axon and elongate the axon to reach its target. It is known that actin filaments and their dynamic interaction with microtubules within growth cones play important roles in inducing axon extension. However, it remains unclear how cytoskeletal dynamics is controlled in growth cones. In this study, we report that Rufy3, a RUN domain-containing protein, is a neuron-specific and actin filament-relevant protein. We find that the appropriate expression of Rufy3 in mouse hippocampal neurons is required for the development of a single axon and axon growth. Our results show that Rufy3 specifically interacts with actin filament-binding proteins, such as Fascin, and colocalizes with Fascin in growth cones. Knockdown of Rufy3 impairs the distribution of Fascin and actin filaments, accompanied by an increased proportion of neurons with multiple axons and a decrease in the axon length. Therefore, Rufy3 may be particularly important for neuronal axon elongation by interacting with Fascin to control actin filament organization in axonal growth cones. We propose that Rufy3 may control mouse neuron axon development through its specific interaction with Fascin and Drebrin. Over-expression of Rufy3 (Rufy3 OE) leads to longer axons and expands the distribution of Drebrin to almost the entire growth cone. In contrast, knockdown of Rufy3 (Rufy3 RNAi) results in shortened axons and enhanced the percentage of mutipolar neurons. Moreover, silencing of Rufy3 reduces and restricts the expression of Fascin and F-actin to the edge of the growth cone. These findings provide new insights into the molecular regulation of axonal outgrowth and cell polarization in neurons.

Collaborative Roles for RAC1, ERM Proteins and PTEN During Adult Sensory Axon Regeneration.

The role of local of growth cone (GC) manipulation in adult regenerative systems is largely unexplored despite substantial translational importance. Here we investigated collaboration among Rac1 GTPase, its partnering ERM proteins and PTEN in adult sensory neurons and adult nerve regeneration. We confirmed expression of both Rac1 and ERM in adults and noted substantial impacts on neurite outgrowth in naïve and pre-injured adult sensory neurons. PTEN inhibition added to this outgrowth. Rac1 activation acted directly on adult GCs facilitating both attractive turning and advancement. In vivo regeneration indices including electrophysiological recovery, return of sensation, walking, repopulation of myelinated axons and reinnervation of the target epidermis indicated benefits of local Rac1 activation. These indices suggested maximal GC activation whereas local PTEN inhibition offered only limited added improvement. Our findings provide support for the concept of manipulating adult GCs, by emphasizing local Rac1 activation in directing therapy for nerve repair.

Non-canonical retinoid signaling in neural development, regeneration and synaptic function.

Canonical retinoid signaling via nuclear receptors and gene regulation is critical for the initiation of developmental processes such as cellular differentiation, patterning and neurite outgrowth, but also mediates nerve regeneration and synaptic functions in adult nervous systems. In addition to canonical transcriptional regulation, retinoids also exert rapid effects, and there are now multiple lines of evidence supporting non-canonical retinoid actions outside of the nucleus, including in dendrites and axons. Together, canonical and non-canonical retinoid signaling provide the precise temporal and spatial control necessary to achieve the fine cellular coordination required for proper nervous system function. Here, we examine and discuss the evidence supporting non-canonical actions of retinoids in neural development and regeneration as well as synaptic function, including a review of the proposed molecular mechanisms involved.

CKAP5 enables formation of persistent actin bundles templated by dynamically instable microtubules.

Cytoskeletal rearrangements and crosstalk between microtubules and actin filaments are vital for living organisms. Recently, an abundantly present microtubule polymerase, CKAP5 (XMAP215 homolog), has been reported to play a role in mediating crosstalk between microtubules and actin filaments in the neuronal growth cones. However, the molecular mechanism of this process is unknown. Here, we demonstrate, in a reconstituted system, that CKAP5 enables the formation of persistent actin bundles templated by dynamically instable microtubules. We explain the templating by the difference in CKAP5 binding to microtubules and actin filaments. Binding to the microtubule lattice with higher affinity, CKAP5 enables the formation of actin bundles exclusively on the microtubule lattice, at CKAP5 concentrations insufficient to support any actin bundling in the absence of microtubules. Strikingly, when the microtubules depolymerize, actin bundles prevail at the positions predetermined by the microtubules. We propose that the local abundance of available CKAP5-binding sites in actin bundles allows the retention of CKAP5, resulting in persisting actin bundles. In line with our observations, we found that reducing CKAP5 levels in vivo results in a decrease in actin-microtubule co-localization in growth cones and specifically decreases actin intensity at microtubule plus ends. This readily suggests a mechanism explaining how exploratory microtubules set the positions of actin bundles, for example, in cytoskeleton-rich neuronal growth cones.

Visualizing Cofilin-Actin Filaments by Immunofluorescence and CryoEM: Essential Steps for Observing Cofilactin in Cells.

Fluorescence microscopy of cytoskeletal proteins in situ using immunolabeling, fluorescent reagents, or expression of tagged proteins has been a common practice for decades but often with too little regard for what might not be visualized. This is especially true for assembled filamentous actin (F-actin), for which binding of fluorescently labeled phalloidin is taken as the gold standard for its quantification even though it is well known that F-actin saturated with cofilin (cofilactin) binds neither fluorescently labeled phalloidin nor genetically encoded F-actin reporters, such as LifeAct. Here, using expressed fluorescent cofilactin reporters, we show that cofilactin is the major component of some actin-containing structures in both normal and stressed neurons and present various fixation, permeabilization, and cryo-preservation methods for optimizing its observation.

Imaging the structural organization of chemical elements in growth cones of developing hippocampal neurons.

During neurodevelopment, neurons form growth cones, F-actin rich extensions located at the distal end of the neurites. Growth cones allow dendrites and axons to build synaptic connections through a process of neurite guidance whose mechanisms have not been fully elucidated. Calcium is an important element in this process by inducing F-actin reorganization. We hypothesized that other biologically active elements might be involved in the growth cone-mediated neurite guidance mechanisms. We performed super resolution and confocal microscopy of F-actin, followed by synchrotron X-ray fluorescence microscopy of phosphorous, sulfur, chlorine, potassium, calcium, iron and zinc on growth cones from primary rat hippocampal neurons. We identified two main patterns of element organization. First, active growth cones presenting an asymmetric distribution of Ca co-localized with the cytoskeleton protein F-actin. In active growth cones, we found that the distributions of P, S, Cl, K, and Zn are correlated with Ca. This correlation is lost in the second pattern, quiescent growth cones, exhibiting a spread elemental distribution. These results suggest that Ca is not the only element required in the F-actin rich active regions of growth cones. In addition, highly concentrated Fe spots of submicrometer size were observed in calcium-rich areas of active growth cones. These results reveal the need for biological active elements in growth cones during neural development and may help explain why early life deficiencies of elements, such as Fe or Zn, induce learning and memory deficits in children.

Förster resonance energy transfer efficiency of the vinculin tension sensor in cultured primary cortical neuronal growth cones.

Significance: Interaction of neurons with their extracellular environment and the mechanical forces at focal adhesions and synaptic junctions play important roles in neuronal development. Aim: To advance studies of mechanotransduction, we demonstrate the use of the vinculin tension sensor (VinTS) in primary cultures of cortical neurons. VinTS consists of TS module (TSMod), a Förster resonance energy transfer (FRET)-based tension sensor, inserted between vinculin's head and tail. FRET efficiency decreases with increased tension across vinculin. Approach: Primary cortical neurons cultured on glass coverslips coated with poly-d-lysine and laminin were transfected with plasmids encoding untargeted TSMod, VinTS, or tail-less vinculinTS (VinTL) lacking the actin-binding domain. The neurons were imaged between day in vitro (DIV) 5 to 8. We detail the image processing steps for calculation of FRET efficiency and use this system to investigate the expression and FRET efficiency of VinTS in growth cones. Results: The distribution of fluorescent constructs was similar within growth cones at DIV 5 to 8. The mean FRET efficiency of TSMod ( 28.5 ± 3.6 % ) in growth cones was higher than the mean FRET efficiency of VinTS ( 24.6 ± 2 % ) and VinTL ( 25.8 ± 1.8 % ) ( p < 10 - 6 ). While small, the difference between the FRET efficiency of VinTS and VinTL was statistically significant ( p < 10 - 3 ), suggesting that vinculin is under low tension in growth cones. Two-hour treatment with the Rho-associated kinase inhibitor Y-27632 did not affect the mean FRET efficiency. Growth cones exhibited dynamic changes in morphology as observed by time-lapse imaging. VinTS FRET efficiency showed greater variance than TSMod FRET efficiency as a function of time, suggesting a greater dependence of VinTS FRET efficiency on growth cone dynamics compared with TSMod. Conclusions: The results demonstrate the feasibility of using VinTS to probe the function of vinculin in neuronal growth cones and provide a foundation for studies of mechanotransduction in neurons using this tension probe.

A Co-purification Method for Efficient Production and Src Kinase-mediated Phosphorylation of Aplysia Cortactin.

Cortactin is an actin-binding protein that regulates processes like cell migration, endocytosis, and tumor cell metastasis. Although cortactin is associated with actin-cytoskeletal dynamics in non-neuronal cells and cell-free systems, the exact mechanisms underlying its fundamental roles in neuronal growth cones are not fully explored. Recent reports show that Aplysia Src2 tyrosine kinase induces phosphorylation of cortactin as a mechanism to control lamellipodia protrusion and filopodia formation in cultured Aplysia bag cell neurons ( He et al., 2015 ; Ren et al., 2019 ). In order to provide in vitro evidence for Src2-mediated phosphorylation of cortactin, we developed a robust and cost-effective method for the efficient expression and purification of Aplysia cortactin and Src2 kinase that can be used for biochemical studies including phosphorylation assays. By co-purifying cortactin and Src kinase with a phosphatase (YopH) from Yersinia enterocolitica, we eliminated the problem of non-specific phosphorylation of induced proteins by bacterial kinases and also reduced costs by bypassing the need for commercial enzymatic treatments. This protocol is reproducible and can be modified to produce homogenous non-phosphorylated proteins during recombinant protein expression in Escherichia coli.

Preparation of Herpes Simplex Virus-Infected Primary Neurons for Transmission Electron Microscopy.

Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus-host cell interaction.While there are many sample preparation techniques for TEM, conventional processing using chemical fixation and resin embedding remains a useful technique, available in virtually all EM laboratories, for studying virus/cell ultrastructure. In this chapter, we describe the preparation of herpes simplex virus infected primary neurons, grown on plastic coverslips, to allow for sectioning of neurons and axons in their growth plane. This technique allows for TEM examination of cell bodies, axons, growth cones and varicosities, providing powerful insights into virus-cell interaction.