Bioactive diamond scaffolds support neuronal survival and axonal growth

Activity Regulates Positive and Negative Neurotrophin-Derived Signals to Determine Axon Competition

In the adult goldfish visual pathway, expression of the neuronal intermediate filament (nIF) protein plasticin is restricted to differentiating retinal ganglion cells (RGCs) at the margin of the retina. Following optic nerve injury, plasticin expression is elevated transiently in all RGCs coincident with the early stages of axon regeneration. These results suggest that plasticin may be expressed throughout the nervous system during the early stages of axonogenesis. To test this hypothesis, we analyzed plasticin expression during zebrafish (Danio rerio) neuronal development. By using immunocytochemistry and in situ hybridization, we found that plasticin is expressed in restricted subsets of early zebrafish neurons. Expression coincides with axon outgrowth in projection neurons that pioneer distinct axon tracts in the embryo. Plasticin is expressed first in trigeminal, Rohon-Beard, and posterior lateral line ganglia neurons, which are among the earliest neurons to initiate axonogenesis in zebrafish. Plasticin is expressed also in reticulospinal neurons and in caudal primary motoneurons. Together, these neurons establish the first behavioral responses in the embryo. Plasticin expression also coincides with initial RGC axonogenesis and progressively decreases after RGC axons reach the tectum. At later developmental stages, plasticin is expressed in a subset of the cranial nerves. The majority of plasticin-positive neurons are within or project axons to the peripheral nervous system. Our results suggest that plasticin subserves the changing requirements for plasticity and stability during axonal outgrowth in neurons that project long axons.

Astrocytes occupy a central role in central nervous system (CNS) function. In particular astyrocytes can support neurite growth, in part, by release of diffusable factors. We therefore performed biochemical analysis of astrocyte conditioned medium to examine possible mechanisms of astrocyte mediated axon and dendrite growth in the mammalian CNS. Culture medium was conditioned on purified astrocyte monolayers derived from P3 rat cerebral cortex or on fibroblasts. Conditioned medium (CM) was subject to protein denaturation, molecular weight fractionation, and heparin affinity chromatography. E18 mouse cerebral cortical neurons were then cultured in the various media or directly on astrocyte monolayers and axon and dendrite growth from 50 neurons in each condition quantified after 3 DIV using double-labeled immunohistochemical techniques. Axon and dendrite growth was supported by astrocyte CM and both were significantly greater than process growth from neurons incubated in fibroblast CM. Protein denaturation significantly reduced astrocyte CM support of axon and dendrite growth. Following ultrafiltration and dialysis dendrite and axon growth was observed in the molecular weight fraction between 10 and 100 kDa. Axon growth also was observed in the CM molecular weight fraction greater than 100 kDa. Conditioned medium was eluted on a heparin column; when the bound fragment was reconstituted in chemically defined medium extensive dendrite and axon growth was observed. Since fibroblast growth factor (FGF) has these biochemical characteristics we added anti-bFGF neutralizing antibodies to astrocyte monolayers or CM; this significantly reduced astrocyte support of process growth. By contrast, the addition of heparin, which helps activate FGF receptors, to astrocyte CM further enhanced process growth. Western blot analysis confirmed that bFGF was present in astrocyte CM. We then examined axon and dendrite growth from cortical neurons after the addition of various growth factors to chemically defined medium. Axon and dendrite growth, similar to that found in astrocyte CM was observed after the addition of bFGF or aFGF. Astrocyte support of cerebral cortical neuron axon and dendrite growth in vitro may be explained, in part, by FGF release. [Neurol Res 2002; 24: 81-92]

Transcriptional Regulation of Neuronal Polarity and Morphogenesis in the Mammalian Brain

Vector systems for the regulated and reversible expression of therapeutic genes are likely to improve the safety and efficacy of gene therapy for medical disease. In the present study, we investigated whether the expression of genes transferred into the central nervous system by ex vivo gene therapy can be regulated in vivo leading to controlled neuronal survival and axonal growth. Primary rat fibroblasts were transfected with a retrovirus containing a tetracycline responsive promoter for the expression of the neurotrophin nerve growth factor (NGF) or green fluorescent protein as a control (GFP). After lesions of basal forebrain cholinergic neurons, NGF-mediated neuronal rescue and axonal growth could be completely controlled over a 2-week period by the addition or removal of the tetracycline modulator doxycycline in the animals' drinking water. Further, continued expression of the reporter gene GFP could be reliably and repeatedly turned on and off in the injured CNS for at least 3 months post-grafting, the longest time point investigated. These data constitute the first report of regulated neuronal rescue and axonal growth by controlled neurotrophin gene delivery and long-term, regulated expression using ex vivo CNS gene therapy.

cAMP signaling is known to be critical in neuronal survival and axon growth. Increasingly the subcellular compartmentation of cAMP signaling has been appreciated, but outside of dendritic synaptic regulation, few cAMP compartments have been defined in terms of molecular composition or function in neurons. Specificity in cAMP signaling is conferred in large part by A-kinase anchoring proteins (AKAPs) that localize protein kinase A and other signaling enzymes to discrete intracellular compartments. We now reveal that cAMP signaling within a perinuclear neuronal compartment organized by the large multivalent scaffold protein mAKAPα promotes neuronal survival and axon growth. mAKAPα signalosome function is explored using new molecular tools designed to specifically alter local cAMP levels as studied by live-cell FRET imaging. In addition, enhancement of mAKAPα-associated cAMP signaling by isoform-specific displacement of bound phosphodiesterase is demonstrated to increase retinal ganglion cell survival in vivo in mice of both sexes following optic nerve crush injury. These findings define a novel neuronal compartment that confers cAMP regulation of neuroprotection and axon growth and that may be therapeutically targeted in disease.SIGNIFICANCE STATEMENT cAMP is a second messenger responsible for the regulation of diverse cellular processes including neuronal neurite extension and survival following injury. Signal transduction by cAMP is highly compartmentalized in large part because of the formation of discrete, localized multimolecular signaling complexes by A-kinase anchoring proteins. Although the concept of cAMP compartmentation is well established, the function and identity of these compartments remain poorly understood in neurons. In this study, we provide evidence for a neuronal perinuclear cAMP compartment organized by the scaffold protein mAKAPα that is necessary and sufficient for the induction of neurite outgrowth in vitro and for the survival of retinal ganglion cells in vivo following optic nerve injury.

NELL2 Function in Axon Development of Hippocampal Neurons

Functional decline in Parkinson's disease (PD) is the cumulative result of regulatory alterations affecting large numbers of genes involved in various aspects of neuronal maintenance and function, and it has recently emerged that axonal degeneration plays a key role. Central to the alterations in gene expression that underlie this failure of axon maintenance, is the regulation of gene transcription by enzymes known as histone deacetylases (HDAC) which promote the de‐acetylation of histone proteins to inhibit transcription. alpha‐synuclein, a key protein that accumulates in the brains of patients with the disease, binds histone proteins in vitro & in vivo, and nuclear a‐synuclein reduces acetylated histone 3 (AcH3) levels. However while these molecular insights are important, how they regulate the neuronal structure and axonal growth in neurons affected by Parkinson's, and the relevance of this cell biology approach to the human brain is unknown.Here we used a network approach to identify all genes that had a significant positive (r+) or negative (r−) correlation with the expression of all HDACs using microarray data from the human substantia nigra (SN; GSE 60863). We then conducted a detailed enrichment analysis to identify biological pathways that were over‐represented and may be linked to Parkinson's Disease. These analyses showed that Class‐II but not Class‐I HDACs were significantly correlated with functional pathways linked to Parkinson's Disease (p<0.0001). These included genes linked to familial Parkinson's Disease and oxidative phosphorylation, including those associated with mitochondrial complex I.To link these data with changes in neuronal structure, we next examined the effects of class specific HDAC inhibitors on neuronal structure by examining a range of HDAC inhibitors for their ability to promote axonal growth and branching in dopaminergic and sympathetic neurons which are the two main cell types that undergo progressive axonal degeneration in Parkinson's Disease. We found that a MC1568 (a class II inhibitor) but not MS275 (a class I inhibitor) promoted significant increases in axon growth and branching (p<0.01). Therefore we further examined the therapeutic potential of MC1568, and found it protected against the detrimental effects of MPP+‐ and alpha‐synuclein induced axonal degeneration in a models of Parkinson's disease.This study therefore provides new insights into the regulator mechanisms that control axonal growth in neurons affects by Parkinson's, and highlights the potential of using this approach to identify therapeutic targets for neuroprotection and axonal regeneration.Support or Funding InformationThis work was funded by a Career Development Award from Science Foundation Ireland under the grant number 15/CDA/3498.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular mechanisms of neuronal specification

Metabotropic glutamate receptor 5 (mGluR5) is extensively involved in neural survival, differentiation, dendritic morphology, synaptic plasticity, and neural circuit formation. However, little is known about its role in neuronal polarization and axon outgrowth. In this study, we applied the selective agonist (RS)-2-chloro-5-hydroxyphenylglycine sodium salt and antagonist 3-[(2-methyl-4-thaizolyl) ethynyl] pyridine (MTEP) of mGluR5 to the cultured hippocampal neurons to observe the neuronal polarization and axon outgrowth, and further explored the possible intracellular signal transduction pathway. The results demonstrated that MTEP administration significantly attenuates the proportion of polarized neurons and the length of the axon, indicated by SMI312 (an axon marker) and Tuj-1 (a marker of all the neurites) double-labeling immunofluorescence. Western blot analysis showed that MTEP administration also inhibited the activation of AKT and nuclear translocation of nuclear factor-κB (NF-κB) p65, and decreased the phosphorylation of p65 as well. Furthermore, Akt inhibitor LY294002 treatment resulted in neuronal polarization delay and axon outgrowth retardation, while suppressing the phosphorylation and nuclear translocation of p65. We concluded that mGluR5 could regulate neuronal polarity and axon outgrowth during the morphological differentiation of rat developing neurons, and the intracellular signaling pathway of Akt-NF-κB might be involved in the action of mGluR5. © 2016 Wiley Periodicals, Inc.

The post-ganglionic sympathetic neurons play an important role in modulating visceral functions and maintaining homeostasis through complex and reproducible axonal and dendritic connections between individual neurons and with their target tissues. Disruptions in these connections and in sympathetic nervous system function are observed in several neurological, cardiac and immune-related disorders, which underscores the need for understanding the mechanisms underlying neuronal polarity, axonal growth and dendritic growth in these neurons. The goals of this chapter are to explore our current understanding of the various growth factors, their signaling pathways, downstream effectors and interplay between these pathways to regulate different stages of axonal and dendritic growth in sympathetic neurons.

The growth of axons is an intricately regulated process involving intracellular signaling cascades and gene transcription. We had previously shown that the stimulus-dependent transcription factor, serum response factor (SRF), plays a critical role in regulating axon growth in the mammalian brain. However, the molecular mechanisms underlying SRF-dependent axon growth remains unknown. Here we report that SRF is phosphorylated and activated by GSK-3 to promote axon outgrowth in mouse hippocampal neurons. GSK-3 binds to and directly phosphorylates SRF on a highly conserved serine residue. This serine phosphorylation is necessary for SRF activity and for its interaction with MKL-family cofactors, MKL1 and MKL2, but not with TCF-family cofactor, ELK-1. Axonal growth deficits caused by GSK-3 inhibition could be rescued by expression of a constitutively active SRF. The SRF target gene and actin-binding protein, vinculin, is sufficient to overcome the axonal growth deficits of SRF-deficient and GSK-3-inhibited neurons. Furthermore, short hairpin RNA-mediated knockdown of vinculin also attenuated axonal growth. Thus, our findings reveal a novel phosphorylation and activation of SRF by GSK-3 that is critical for SRF-dependent axon growth in mammalian central neurons.

The receptor deleted in colorectal cancer (DCC) mediates the attraction of growing axons to netrin-1 during brain development. In response to netrin-1 stimulation, DCC becomes a signaling platform to recruit proteins that promote axon outgrowth and guidance. The Ras GTPase-activating protein (GAP) p120RasGAP inhibits Ras activity and mediates neurite retraction and growth cone collapse in response to repulsive guidance cues. Here we show an interaction between p120RasGAP and DCC that positively regulates netrin-1-mediated axon outgrowth and guidance in embryonic cortical neurons. In response to netrin-1, p120RasGAP is recruited to DCC in growth cones and forms a multiprotein complex with focal adhesion kinase and ERK. We found that Ras/ERK activities are elevated aberrantly in p120RasGAP-deficient neurons. Moreover, the expression of p120RasGAP Src homology 2 (SH2)-SH3-SH2 domains, which interact with the C-terminal tail of DCC, is sufficient to restore netrin-1-dependent axon outgrowth in p120RasGAP-deficient neurons. We provide a novel mechanism that exploits the scaffolding properties of the N terminus of p120RasGAP to tightly regulate netrin-1/DCC-dependent axon outgrowth and guidance.

Essential Roles for GSK-3s and GSK-3-Primed Substrates in Neurotrophin-Induced and Hippocampal Axon Growth

The canonical Wnt/β-catenin pathway is a highly conserved signaling cascade that plays critical roles during embryogenesis. Wnt ligands regulate axonal extension, growth cone guidance and synaptogenesis throughout the developing central nervous system (CNS). Recently, studies in mammalian and fish model systems have demonstrated that Wnt/β-catenin signaling also promotes axonal regeneration in the adult optic nerve and spinal cord after injury, raising the possibility that Wnt could be developed as a therapeutic strategy. In this review, we summarize experimental evidence that reveals novel roles for Wnt signaling in the injured CNS, and discuss possible mechanisms by which Wnt ligands could overcome molecular barriers inhibiting axonal growth to promote regeneration. A central challenge in the neuroscience field is developing therapeutic strategies that induce robust axonal regeneration. Although adult axons have the capacity to respond to axonal guidance molecules after injury, there are several major obstacles for axonal growth, including extensive neuronal death, glial scars at the injury site, and lack of axonal guidance signals. Research in rodents demonstrated that activation of Wnt/β-catenin signaling in retinal neurons and radial glia induced neuronal survival and axonal growth, but that activation within reactive glia at the injury site promoted proliferation and glial scar formation. Studies in zebrafish spinal cord injury models confirm an axonal regenerative role for Wnt/β-catenin signaling and identified the cell types responsible. Additionally, in vitro and in vivo studies demonstrated that Wnt induces axonal and neurite growth through transcription-dependent effects of its central mediator β-catenin, potentially by inducing regeneration-promoting genes. Canonical Wnt signaling may also function through transcription-independent interactions of β-catenin with cytoskeletal elements, which could stabilize growing axons and control growth cone movement. Therefore, these studies suggest that Wnt-induced pathways responsible for regulating axonal growth during embryogenesis could be repurposed to promote axonal growth after injury.