Neuronal Growth Cones Research Articles

Rapid Assembly of Presynaptic Materials behind the Growth Cone in Dopaminergic Neurons Is Mediated by Precise Regulation of Axonal Transport

SUMMARYThe proper assembly of neural circuits depends on the process of synaptogenesis, or the formation of synapses between partner neurons. Using the dopaminergic PDE neurons in C. elegans, we developed an in vivo system to study the earliest steps of the formation of en passant presynaptic specializations behind an extending growth cone. We find that presynaptic materials coalesce into puncta in as little as a few minutes and that both synaptic vesicle (SV) and active zone (AZ) proteins arrive nearly simultaneously at the nascent sites of synapse formation. We show that precise regulation of UNC-104/Kinesin-3 determines the distribution of SV proteins along the axon. The localization of AZ proteins to en passant puncta, however, is largely independent of the major axonal kinesins: UNC-104/Kinesin-3 and UNC-116/ Kinesin-1. Moreover, AZ proteins play a crucial role in recruiting and tethering SV precursors (SVPs).

Neuronal Growth Cone Size-Dependent and -Independent Parameters of Microtubule Polymerization.

Migration and pathfinding of neuronal growth cones during neurite extension is critically dependent on dynamic microtubules. In this study we sought to determine, which aspects of microtubule polymerization relate to growth cone morphology and migratory characteristics. We conducted a multiscale quantitative microscopy analysis using automated tracking of microtubule plus ends in migrating growth cones of cultured murine dorsal root ganglion (DRG) neurons. Notably, this comprehensive analysis failed to identify any changes in microtubule polymerization parameters that were specifically associated with spontaneous extension vs. retraction of growth cones. This suggests that microtubule dynamicity is a basic mechanism that does not determine the polarity of growth cone response but can be exploited to accommodate diverse growth cone behaviors. At the same time, we found a correlation between growth cone size and basic parameters of microtubule polymerization including the density of growing microtubule plus ends and rate and duration of microtubule growth. A similar correlation was observed in growth cones of neurons lacking the microtubule-associated protein MAP1B. However, MAP1B-null growth cones, which are deficient in growth cone migration and steering, displayed an overall reduction in microtubule dynamicity. Our results highlight the importance of taking growth cone size into account when evaluating the influence on growth cone microtubule dynamics of different substrata, guidance factors or genetic manipulations which all can change growth cone morphology and size. The type of large scale multiparametric analysis performed here can help to separate direct effects that these perturbations might have on microtubule dynamics from indirect effects resulting from perturbation-induced changes in growth cone size.

CHP1 reduction ameliorates spinal muscular atrophy pathology by restoring calcineurin activity and endocytosis.

Autosomal recessive spinal muscular atrophy (SMA), the leading genetic cause of infant lethality, is caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. SMA disease severity inversely correlates with the number of SMN2 copies, which in contrast to SMN1, mainly produce aberrantly spliced transcripts. Recently, the first SMA therapy based on antisense oligonucleotides correcting SMN2 splicing, namely SPINRAZATM, has been approved. Nevertheless, in type I SMA-affected individuals-representing 60% of SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong. Plastin 3 (PLS3) and neurocalcin delta (NCALD) are two SMN-independent protective modifiers identified in humans and proved to be effective across various SMA animal models. Both PLS3 overexpression and NCALD downregulation protect against SMA by restoring impaired endocytosis; however, the exact mechanism of this protection is largely unknown. Here, we identified calcineurin-like EF-hand protein 1 (CHP1) as a novel PLS3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and PLS3. Although CHP1 is ubiquitously present, it is particularly abundant in the central nervous system and at SMA-relevant sites including motor neuron growth cones and neuromuscular junctions. Strikingly, we found elevated CHP1 levels in SMA mice. Congruently, CHP1 downregulation restored impaired axonal growth in Smn-depleted NSC34 motor neuron-like cells, SMA zebrafish and primary murine SMA motor neurons. Most importantly, subcutaneous injection of low-dose SMN antisense oligonucleotide in pre-symptomatic mice doubled the survival rate of severely-affected SMA mice, while additional CHP1 reduction by genetic modification prolonged survival further by 1.6-fold. Moreover, CHP1 reduction further ameliorated SMA disease hallmarks including electrophysiological defects, smaller neuromuscular junction size, impaired maturity of neuromuscular junctions and smaller muscle fibre size compared to low-dose SMN antisense oligonucleotide alone. In NSC34 cells, Chp1 knockdown tripled macropinocytosis whereas clathrin-mediated endocytosis remained unaffected. Importantly, Chp1 knockdown restored macropinocytosis in Smn-depleted cells by elevating calcineurin phosphatase activity. CHP1 is an inhibitor of calcineurin, which collectively dephosphorylates proteins involved in endocytosis, and is therefore crucial in synaptic vesicle endocytosis. Indeed, we found marked hyperphosphorylation of dynamin 1 in SMA motor neurons, which was restored to control level by the heterozygous Chp1 mutant allele. Taken together, we show that CHP1 is a novel SMA modifier that directly interacts with PLS3, and that CHP1 reduction ameliorates SMA pathology by counteracting impaired endocytosis. Most importantly, we demonstrate that CHP1 reduction is a promising SMN-independent therapeutic target for a combinatorial SMA therapy.

HEAVY METAL POLLUTANT-INDUCED CYTOTOXICITY INVOLVES PERTURBATIONS OF MITOCHONDRIAL FUNCTION

Abstract BACKGROUND Environmental pollutants like heavy metals pose a tremendous risk to both animal and human health though the mechanisms underlying their cytotoxic actions at the cellular level remain largely unknown. The freshwater mollusc Lymnaea stagnalis carries hemocynin as its oxygen carrier whereby iron is replaced with copper. As such, this species is highly sensitive to its environmental copper contents and has been used as an important indicator of water quality. Studies have shown that miniscule levels of heavy metals (Pb, Ni, Cu, Co) dissolved in aqueous environments lead to detrimental effects on many of the Lymanea’s vital functions – including respiration and cardiac functions. OBJECTIVES The objective was to determine how heavy metal pulliutants target the cell and its vital functions and if the detirioration of cell motility and viability is a result of the reduced functioning and potential of the cells mitochondria. DESIGN/METHODS We tested the effects of Cu (EC20 as low as 1.8 ug L-1) on isolated blood cells (hemocytes) and neurons from Lymnaea. We sought to determine whether Cu2+ affected cellular viability, motility and neuronal growth cone movements involving cytoskeletal proteins such as actin and tubulin. Cells from the brain were harvested and plated on sigma coated dishes and left to incubate for 3 days to adjust to the environement. They were then stained with mitortracker dyes for mitochondrial potential and movement. The results were captured through live cell imaging and were later analyzed using neurite tracer. We demonstrate the effects of Cu on single mitochondrial movements, structure and function. RESULTS This study provides the first direct evidence that heavy metals such as Cu are indeed cytotoxic and that its detrimental effects on animal health likely involve perturbations of mitochondrial structure and function. Mitochondrial accumulation within the cell started detirioraiting within the first hour of live cell imaging. The mitochondria within the tested cells also started chainging how they cluster within an expanding growth cone. Mitochondrial density decreased substantially and clustered mitohochondira became more fragmeneted which is a positive indication of mitochndrial malfunction. The mitochondrial potential dropped substantially too, indicating that the mitochondria were unable to produce energy like they are suppose to in normal conditions. Finally, this also allowed us to show why heavy metal use in the dentistry profession and other medical professions where heavy metals are used can be linked to cell apoptosis and mitochondrial degradation. CONCLUSION Heavy metals indeed cause malfunction within the cells mitochondria and the reduced functioning disables the cells from fulfilling their associated functions. This was observed through the reduction of breathing, mastication, movement, and neuronal firing of the Lymnaea, which shows a direct realtionship to degraded mitochondria within these cells. Similar results were seen within the hemocytes, which demonstrates that their function of circulating and delivering oxygen was also impaired due to faulty mitochondria.

BDNF Reduces eEF2 Phosphorylation and Enhances Novel Protein Synthesis in the Growth Cones of Dorsal Root Ganglia Neurons.

The local translation, which is regulated by extracellular stimuli such as guidance molecules, in growth cones of neurons provides a molecular mechanism for axonal development. In this study, we performed immunocytochemistry together with atomic force microscopy to investigate the localization of ribosomal proteins in the growth cones of rat dorsal root ganglion (DRG) neurons. The immunoreactivity of ribosomal protein P0/1/2 and S6, and novel protein synthesis were observed in the central, sterically bulky region of growth cones. Brain derived neurotrophic factor (BDNF) reduced the eEF2 phosphorylation, indicating its activation, and enhanced protein synthesis within 30min. The effects of BDNF were completely inhibited by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR). These results indicated that BDNF rapidly activates translation and enhances novel protein synthesis in growth cones of DRG though the mTOR signaling.

Axon growth regulation by a bistable molecular switch

For the brain to function properly, its neurons must make the right connections during neural development. A key aspect of this process is the tight regulation of axon growth as axons navigate towards their targets. Neuronal growth cones at the tips of developing axons switch between growth and paused states during axonal pathfinding, and this switching behaviour determines the heterogeneous axon growth rates observed during brain development. The mechanisms controlling this switching behaviour, however, remain largely unknown. Here, using mathematical modelling, we predict that the molecular interaction network involved in axon growth can exhibit bistability, with one state representing a fast-growing growth cone state and the other a paused growth cone state. Owing to stochastic effects, even in an unchanging environment, model growth cones reversibly switch between growth and paused states. Our model further predicts that environmental signals could regulate axon growth rate by controlling the rates of switching between the two states. Our study presents a new conceptual understanding of growth cone switching behaviour, and suggests that axon guidance may be controlled by both cell-extrinsic factors and cell-intrinsic growth regulatory mechanisms.

Copper Binding Regulates Cellular Prion Protein Function

The cellular prion protein (PrPC), mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neuritogenesis. By combining genomic approaches, cellular assays and focal stimulation technique, we have explored the molecular mechanism underlining PrPC function as a signaling molecule in neuritogenesis. Several recombinant prion protein (recPrP) mutants were obtained to treat primary hippocampal cultures in bulk or exposed near the hippocampal growth cones (GC) of single neurons in a local stimulation manner. While focal stimulation of GC with wild‐type recPrP induced neurite outgrowth and rapid GC turning towards the source, N‐terminal mutants fail to support this function.In particular, the copper‐binding sites mutants present at the N‐terminus of PrPC are toxic to neurons indicating this region being crucial for the function of the protein. Mutants of recPrP including a key mutation for prion conversion H95Y (Giachin, Mai et al. 2015) or a GSS‐linked mutation abolish the function on neuritogenesis. Altogether, our findings indicate the functional regions for PrPC involved in neuritogenesis, suggest a potential link between loss‐of‐function of the protein and disease initiation.Support or Funding InformationFondo per gli Investimenti della Ricerca di Base (FIRB) program project (grant number RBAP11FRE9_001 to GL) from Ministero dell'Istruzione, dell'Università e della Ricerca(MIUR), ItalyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CB1 BUT NOT CB2 CANNABINOID RECEPTOR INCREASES NEURITE EXTENSION IN HUMAN NEUROBLASTOMA

Despite neurodegenerative disease affecting more than five million Americans, there is not yet sufficient understanding of the underlying mechanism of disease to cure or even halt progression. Transplanted neurons may represent a potential treatment if they could successfully integrate with host brain tissue. Both CB1 and CB2 receptors are located in neuronal growth cone membranes, implying that cannabinoid signaling may play a role in neurite extension or guidance (Harkany et al. 2007). CB1 cannabinoid signaling has been shown to be active in neuronal extension, yet the differences between CB1R and CB2R signaling remain unclear (Berghuis et al. 2007, Duff et al. 2013). We have created a novel stable overexpression model to investigate the role of cannabinoid signaling in human neuronal extension. We quantified the endogenous production of cannabinoid ligand 2‐Arachidonoyl Glycerol (2‐AG) and its isomer 1‐AG in SHSY5Y human neuroblastoma at an abundance of 4,000 nanograms Total AG per million cells. This AG was likely 2‐AG produced by DAGL enzyme activity because calcium stimulation by Oxotremorine M increased Total AG abundance to 6,000 nanograms per million cells while inhibition of DAGL by THL decreased Total AG abundance to less than 100 nanograms per million cells. We transfected SHSY5Y cells with either CB1 or CB2 receptors, purified and maintained the transfection using geneticin selection, and isolated clonal cell lines that stably overexpress either CB1R (CB1XS) or CB2R (CB2XS). Stable overexpression of cannabinoid receptors did not alter mRNA abundance of the enzymes that synthesize and degrade the endogenous cannabinoids 2‐AG and AEA. Stable overexpression of CB1R but not CB2R increased the extension length from 30 to 200 micrometers per neurite. Our research suggests a role for CB1R but not CB2R mediated cannabinoid signaling in neurite development that may provide insights in development of therapies that support functional integration of transplanted neurons into host tissue.Support or Funding InformationSupport from NIDA grant DA03690 and T32 grant HL091797 is appreciated.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Single-cell nanobiopsy reveals compartmentalization of mRNAs within neuronal cells

In highly polarized cells such as neurons, compartmentalization of mRNA and of local protein synthesis enables remarkably fast, precise, and local responses to external stimuli. These responses are highly important for neuron growth cone guidance, synapse formation, and regeneration following injury. Because an altered spatial distribution of mRNA can result in mental retardation or neurodegenerative diseases, subcellular transcriptome analysis of neurons could be a useful tool for studying these conditions, but current techniques, such as in situ hybridization, bulk microarray, and RNA-Seq, impose tradeoffs between spatial resolution and multiplexing. To obtain a comprehensive analysis of the cell body versus neurite transcriptome from the same neuron, we have recently developed a label-free, single-cell nanobiopsy platform based on scanning ion conductance microscopy that uses electrowetting within a quartz nanopipette to extract cellular material from living cells with minimal disruption of the cellular membrane and milieu. In this study, we used this platform to collect samples from the cell bodies and neurites of human neurons and analyzed the mRNA pool with multiplex RNA sequencing. The minute volume of a nanobiopsy sample allowed us to extract samples from several locations in the same cell and to map the various mRNA species to specific subcellular locations. In addition to previously identified transcripts, we discovered new sets of mRNAs localizing to neurites, including nuclear genes such as Eomes and Hmgb3 In summary, our single-neuron nanobiopsy analysis provides opportunities to improve our understanding of intracellular mRNA transport and local protein composition in neuronal growth, connectivity, and function.

Neuroglobin boosts axon regeneration during ischemic reperfusion via p38 binding and activation depending on oxygen signal

Cerebral ischemia causes severe cell death or injury including axon breakdown or retraction in the brain. Axon regeneration is crucial for the functional recovery of injured neurons or brains after ischemia/reperfusion (I/R); however, this process has been proved extremely difficult in adult brains and there is still no effective therapy for it. Here we reported that neuroglobin (Ngb), a novel oxygen-binding or sensor protein existing predominantly in neurons or brains, functions as a driving factor for axon regeneration during I/R. Ngb was upregulated and accumulated in growth cones of ischemic neurons in primary cultures, rat, and human brains, correlating positively to the elevation of axon-regeneration markers GAP43, neurofilament-200, and Tau-1. Ngb overexpression promoted while Ngb knockdown suppressed axon regeneration as well as GAP43 expression in neurons during oxygen-glucose deprivation/reoxygenation (OGD/Re). By using specific pharmacological inhibitors, we identified p38 MAPK as the major downstream player of Ngb-induced axon regeneration during OGD/Re. Mechanistically, Ngb directly bound to and activated p38 in neurons upon OGD/Re. Serial truncation and point mutation of Ngb revealed that the 7–105 aa fragment of Ngb was required and the oxygen-binding site (His64) of Ngb was the major regulatory site for its p38 interaction/activation. Finally, administration of exogenous TAT-Ngb peptides significantly enhanced axon regeneration in cultured neurons upon OGD/Re. Taken together, Ngb promotes axon regeneration via O2-Ngb-p38-GAP43 signaling during I/R. This novel mechanism suggests potential therapeutic applications of Ngb for ischemic stroke and other related axonopathy.

Spatially Selective Dissection of Signal Transduction in Neurons Grown on NETRIN-1 Printed Nanoarrays via Segmented Fluorescence Fluctuation Analysis

Axonal growth cones extend during neural development in response to precise distributions of extracellular cues. Deleted in colorectal cancer (DCC), a receptor for the chemotropic guidance cue netrin-1, directs F-actin reorganization, and is essential for mammalian neural development. To elucidate how the extracellular distribution of netrin-1 influences the distribution of DCC and F-actin within axonal growth cones, we patterned nanoarrays of substrate bound netrin-1 using lift-off nanocontact printing. The distribution of DCC and F-actin in embryonic rat cortical neuron growth cones was then imaged using total internal reflection fluorescence (TIRF) microscopy. Fluorescence fluctuation analysis via image cross-correlation spectroscopy (ICCS) was applied to extract the molecular density and aggregation state of DCC and F-actin, identifying the fraction of DCC and F-actin colocalizing with the patterned netrin-1 substrate. ICCS measurement of spatially segmented images based on the substrate nanodot patte...

Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

Heterozygous missense mutations in human TUBB3 gene result in a spectrum of brain malformations associated with defects in axon guidance, neuronal migration and differentiation. However, the molecular mechanisms underlying mutation-related axon guidance abnormalities are unclear. Recent studies have shown that netrin-1, a canonical guidance cue, induced the interaction of TUBB3 with the netrin receptor deleted in colorectal cancer (DCC). Furthermore, TUBB3 is required for netrin-1-induced axon outgrowth, branching and pathfinding. Here, we provide evidence that TUBB3 mutations impair netrin/DCC signaling in the developing nervous system. The interaction of DCC with most TUBB3 mutants (eight out of twelve) is significantly reduced compared to the wild-type TUBB3. TUBB3 mutants R262C and A302V exhibit decreased subcellular colocalization with DCC in the growth cones of primary neurons. Netrin-1 increases the interaction of endogenous DCC with wild-type human TUBB3, but not R262C or A302V, in primary neurons. Netrin-1 also increases co-sedimentation of DCC with polymerized microtubules (MTs) in primary neurons expressing the wild-type TUBB3, but not R262C or A302V. Expression of either R262C or A302V not only suppresses netrin-1-induced neurite outgrowth, branching and attraction in vitro, but also causes defects in spinal cord commissural axon (CA) projection and pathfinding in ovo. Our study reveals that missense TUBB3 mutations specifically disrupt netrin/DCC-mediated attractive signaling.

Preparation of Adult Rat Sensory Neuron Cultures and Their Application to Growth Cone Turning Assays.

The directional trajectory of growing peripheral nerve axons in the adult impacts their successful regeneration to denervated target tissues. Misdirected axons in neuromas, injured nerve trunks, or nerves with attempted repair diminish the success of regeneration. The behavior of adult rodent peripheral sensory neurons in vitro, in turn, is helpful in predicting axonal behavior in vivo. Here, we describe the adaptation of embryonic neuron growth cone turning assays, an important technique in developmental neurobiology, to adult rat sensory neurons. With some key modifications, and selection of subtypes of neurons likely to respond to a purported growth factor, short-term responses to molecular gradients can be analyzed using routine dorsal root ganglion neuronal cell culture techniques. The caveats are that short-term turning does not necessarily reflect on the overall tropic impact of a molecule, particularly if it alters growth cones through intra-axonal translation. Similarly, to understand the trajectory of an axon, it must be in a growth mode, such as that associated with preconditioning from previous injury.

Lentivirus Mediating FGF13 Enhances Axon Regeneration after Spinal Cord Injury by Stabilizing Microtubule and Improving Mitochondrial Function.

Fibroblast growth factor 13 (FGF13), a nonsecretory protein of the FGF family, plays a crucial role in developing cortical neurons by stabilizing the microtubule. In previous studies, we showed that regulation of microtubule dynamics was instrumental for both growth cone initiation and for promoting regrowth of injured axon. However, the expression and effect of FGF13 in spinal cord or after spinal cord injury (SCI) remains undefined. Here, we demonstrated a role of FGF13 in regulating microtubule dynamics and in enhancing axon regeneration after SCI. Administration of FGF13 not only promoted neuronal polarization, axon formation, and growth cone initiation in vitro, but it also facilitated functional recovery following SCI. In addition, we found that upregulation of FGF13 in primary cortical neurons was accompanied by enhanced mitochondrial function, which is essential for axon regeneration. Our study has defined a novel mechanism underlying the beneficial effect of FGF13 on axon regeneration, pointing out that FGF13 may serve as a potential candidate for treating SCI or other central nervous system (CNS) injury.

Revealing chiral cell motility by 3D Riesz transform-differential interference contrast microscopy and computational kinematic analysis

Left–right asymmetry is a fundamental feature of body plans, but its formation mechanisms and roles in functional lateralization remain unclear. Accumulating evidence suggests that left–right asymmetry originates in the cellular chirality. However, cell chirality has not yet been quantitatively investigated, mainly due to the absence of appropriate methods. Here we combine 3D Riesz transform-differential interference contrast (RT-DIC) microscopy and computational kinematic analysis to characterize chiral cellular morphology and motility. We reveal that filopodia of neuronal growth cones exhibit 3D left-helical motion with retraction and right-screw rotation. We next apply the methods to amoeba Dictyostelium discoideum and discover right-handed clockwise cell migration on a 2D substrate and right-screw rotation of subcellular protrusions along the radial axis in a 3D substrate. Thus, RT-DIC microscopy and the computational kinematic analysis are useful and versatile tools to reveal the mechanisms of left–right asymmetry formation and the emergence of lateralized functions.

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

BackgroundThere is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location.Methodology/Principal findingsTo determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3’UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate.Conclusion/SignificanceSome intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

Strong contact coupling of neuronal growth cones with height-controlled vertical silicon nanocolumns

In this study, we report that height-controlled vertically etched silicon nano-column arrays (vSNAs) induce strong growth cone-to-substrate coupling and accelerate In vitroneurite development while preserving the essential features of initial neurite formation. Large-scale preparation of vSNAs with flat head morphology enabled the generation of well-controlled topographical stimulation without cellular impalement. A systematic analysis on topography-induced variations on cellular morphology and cytoskeletal dynamics was conducted. In addition, neurite development on the grid-patterned vSNAs exhibited preferential adhesion to the nanostructured region and outgrowth directionality. The arrangement of cytoskeletal proteins and the expression of a focal adhesion complex indicated that a strong coupling existed between the underlying nanocolumns and growth cones. Furthermore, the height-controlled nanocolumn substrates differentially modulated neurite polarization and elongation. Our findings provide an important insight into neuron-nanotopography interactions and their role in cell adhesion and neurite development.

Homodimeric Kinesin-2 KIF3CC Promotes Microtubule Dynamics

KIF3C is one subunit of the functional microtubule-based kinesin-2 KIF3AC motor, an anterograde cargo transporter in neurons. However, KIF3C has also been implicated as an injury-specific kinesin that is a key regulator of axonal growth and regeneration by promoting microtubule dynamics for reorganization at the neuronal growth cone. To test its potential role as a modulator of microtubule dynamics in vitro, an engineered homodimeric KIF3CC was incorporated into a dynamic microtubule assay and examined by total internal reflection fluorescence microscopy. The results reveal that KIF3CC is targeted to the microtubule plus-end, acts as a potent catastrophe factor through an increase in microtubule catastrophe frequency, and does so by elimination of the dependence of the catastrophe rate on microtubule lifetime. Moreover, KIF3CC accelerates the catastrophe rate without altering the microtubule growth rate. Therefore, the ATP-promoted KIF3CC mechanism of catastrophe is different from the well-described catastrophe factors kinesin-13 MCAK and kinesin-8 Kip3/KIF18A. The properties of KIF3CC were not shared by heterodimeric KIF3AC and required the unique KIF3C-specific sequence extension in loop L11 at the microtubule interface. At the microtubule plus-end, the presence of KIF3CC resulted in modulation of the tapered structure typically seen in growing dynamic microtubules to microtubule blunt plus-ends. Overall our results implicate homodimeric KIF3CC as a unique promoter of microtubule catastrophe and substantiate its physiological role in cytoskeletal remodeling.

Patterned semiconductor structures modulate neuronal outgrowth: Implication for the development of a neurobionic interface

Auditory implants stimulate the neurons by broad electrical fields, which leads to a low number of spectral channels. A reduction in the distance between the electrode and the neuronal structures might lead to better electrical transduction. The use of microstructured semiconductors offers a large number of contacts, which could attract neurons and stimulate them individually. To investigate the interaction between neurons and semiconductors, differentiated neuronal precursor cells were cultured on silicon wafers. Different structures were added on the wafers by electron beam lithography, and deep reactive ion etching in different depths (2 and 7 µm). Grooved surfaces guided the neurons and resulted in straight oriented axons, but neuronal outgrowth was impaired by the 7 µm grooves. Within the 7 µm structures, the neuronal cell body was totally encased and the nuclei were deformed from a round to an elliptical shape. On both square and cylindrical structures neuronal bridging could be detected in different forms, either between the tops of the structures or between the bottom and the top. Furthermore, neuronal bridges were established on the lateral part of the structures, and change in direction of neuronal growth was induced by the structure. Finally, it could be shown that neuronal growth cones were particularly attracted by the top of the cylinders, which might allow for the stimulation of neurons via this structure. In conclusion, study results indicate that structured semiconductors can modulate neuronal growth and its direction, offering a novel method for the development of new implants with improved neuronal stimulation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 65-72, 2018.

Arginyltransferase ATE1 is targeted to the neuronal growth cones and regulates neurite outgrowth during brain development

Arginylation is an emerging protein modification mediated by arginyltransferase ATE1, shown to regulate embryogenesis and actin cytoskeleton, however its functions in different physiological systems are not well understood. Here we analyzed the role of ATE1 in brain development and neuronal growth by producing a conditional mouse knockout with Ate1 deletion in the nervous system driven by Nestin promoter (Nes-Ate1 mice). These mice were weaker than wild type, resulting in low postnatal survival rates, and had abnormalities in the brain that suggested defects in neuronal migration. Cultured Ate1 knockout neurons showed a reduction in the neurite outgrowth and the levels of doublecortin and F-actin in the growth cones. In wild type, ATE1 prominently localized to the growth cones, in addition to the cell bodies. Examination of the Ate1 mRNA sequence reveals the existence of putative zipcode-binding sequences involved in mRNA targeting to the cell periphery and local translation at the growth cones. Fluorescence in situ hybridization showed that Ate1 mRNA localized to the tips of the growth cones, likely due to zipcode-mediated targeting, and this localization coincided with spots of localization of arginylated β-actin, which disappeared in the presence of protein synthesis inhibitors. We propose that zipcode-mediated co-targeting of Ate1 and β-actin mRNA leads to localized co-translational arginylation of β-actin that drives the growth cone migration and neurite outgrowth.