Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line.

The epsilon 4 allele of apolipoprotein E (apoE) is a major risk factor for Alzheimer disease, suggesting that apoE may directly influence neurons in the aging brain. Recent data suggest that apoE-containing lipoproteins can influence neurite outgrowth in an isoform-specific fashion. The neuronal mediators of apoE effects have not been clarified. We show here that in a central nervous system-derived neuronal cell line, apoE3 but not apoE4 increases neurite extension. The effect of apoE3 was blocked at low nanomolar concentrations by purified 39-kDa protein that regulates ligand binding to the low density lipoprotein receptor-related protein (LRP). Anti-LRP antibody also completely abolished the neurite-promoting effect of apoE3. Understanding isoform-specific cell biological processes mediated by apoE-LRP interactions in central nervous system neurons may provide insight into Alzheimer disease pathogenesis.

Restriction of intramuscular neurite branching and extension is lost in agrin and rapsyn-deficient mice

The phrenic nerve enters the diaphragm at approximately embryonic day 12.5 (E12.5) in the mouse. The secondary nerve trunk advances along the centre of the diaphragm muscle and extends tertiary branches primarily towards the lateral side during normal embryonic development. In the present study we quantified the intramuscular neurite branching in the most ventral region of the diaphragm at E15.5 and E18.5 in wild-type mice, agrin knock-out mice (KOAG) and rapsyn knock-out mice (KORAP). KOAG and KORAP have decreased muscle contraction due to their inability to maintain/form acetylcholine receptor (AChR) clusters during embryonic development. Heterozygote mothers were anaesthetised via an overdose of Nembutal (30 mg; Boeringer Ingelheim, Ridgefield, CT, USA) and killed via cervical dislocation. There were increases in the number of branches exiting the medial side of the phrenic nerve trunk in KOAG and KORAP compared to wild-type mice, but not on the lateral side at E15.5 and E18.5. However, the number of bifurcations in the periphery significantly increased on both the medial and lateral sides of the diaphragm at E15.5 and E18.5 in KOAG and KORAP compared to control mice. Furthermore, neurites extended further on both the medial and lateral sides of the diaphragm at E15.5 and E18.5 in KOAG and KORAP compared to wild-type mice. Together these results show that the restriction of neurite extension and bifurcations from the secondary nerve trunk is lost in both KOAG and KORAP allowing us the opportunity to investigate the factors that restrict motoneuron behaviour in mammalian muscles.

The motor neurite terminal determines where neuromuscular synapses form in the absence of agrin

Agrin is a proteoglycan secreted by motor neurite terminals that functions to initiate and maintain AChR clusters at the nerve terminal. This led to the theory that neurite terminals decide where neuromuscular synapses form by secreting agrin. However, initiation of AChR clustering occurs in the absence of the innervating motoneuron and in the absence of agrin. In this instance, the muscle, not the nerve, is deciding the location of neuromuscular synapses by drawing neurite terminals towards pre-existing AChR clusters. If this were true, one would expect the initial innervation patterns to be the same in agrin-deficient mice and wild-type mice. To test this we quantified the intramuscular axonal branching and synapse formation in the diaphragm at E14.5 in agrin-deficient mice and wild-type mice. Heterozygote mothers were anaesthetised with Nembutal (30 mg) and killed via cervical dislocation. In the diaphragm, the nerve trunk runs down the centre of the muscle and extends branches primarily toward the lateral side. In agrin-deficient mice however, we found significantly more branches exited the phrenic nerve trunk, branched in the periphery and extended further on the medial side. Moreover, we found that the percentage α-bungarotoxin/synaptophysin colocalisations, markers of pre- and postsynaptic differentiation, respectively, was the same in agrin-deficient mice and wild-type mice. These results show that initial innervation patterns are not the same in agrin-deficient mice and wild-type mice indicating neurite terminals, not muscle, decide the placement of neuromuscular synapses in the absence of agrin.

A comparison of high-content screening versus manual analysis to assay the effects of mesenchymal stem cell-conditioned medium on neurite outgrowth in vitro

Bone marrow mesenchymal stem cells (MSCs) promote nerve growth and functional recovery in animal models of spinal cord injury (SCI) to varying levels. The authors have tested high-content screening to examine the effects of MSC-conditioned medium (MSC-CM) on neurite outgrowth from the human neuroblastoma cell line SH-SY5Y and from explants of chick dorsal root ganglia (DRG). These analyses were compared to previously published methods that involved hand-tracing individual neurites. Both methods demonstrated that MSC-CM promoted neurite outgrowth. Each showed the proportion of SH-SY5Y cells with neurites increased by ~200% in MSC-CM within 48 h, and the number of neurites/SH-SY5Y cells was significantly increased in MSC-CM compared with control medium. For high-content screening, the analysis was performed within minutes, testing multiple samples of MSC-CM and in each case measuring >15,000 SH-SY5Y cells. In contrast, the manual measurement of neurite outgrowth from >200 SH-SY5Y cells in a single sample of MSC-CM took at least 1 h. High-content analysis provided additional measures of increased neurite branching in MSC-CM compared with control medium. MSC-CM was also found to stimulate neurite outgrowth in DRG explants using either method. The application of the high-content analysis was less well optimized for measuring neurite outgrowth from DRG explants than from SH-SY5Y cells.

Neurite outgrowth by the alternatively spliced region of human tenascin-C is mediated by neuronal alpha7beta1 integrin

The region of tenascin-C containing only alternately spliced fibronectin type-III repeat D (fnD) increases neurite outgrowth by itself and also as part of tenascin-C. We previously localized the active site within fnD to an eight amino acid sequence unique to tenascin-C, VFDNFVLK, and showed that the amino acids FD and FV are required for activity. The purpose of this study was to identify the neuronal receptor that interacts with VFDNFVLK and to investigate the hypothesis that FD and FV are important for receptor binding. Function-blocking antibodies against both alpha7 and beta1 integrin subunits were found to abolish VFDNFVLK-mediated process extension from cerebellar granule neurons. VFDNFVLK but not its mutant, VSPNGSLK, induced clustering of neuronal beta1 integrin immunoreactivity. This strongly implicates FD and FV as important structural elements for receptor activation. Moreover, biochemical experiments revealed an association of the alpha7beta1 integrin with tenascin-C peptides containing the VFDNFVLK sequence but not with peptides with alterations in FD and/or FV. These findings are the first to provide evidence that the alpha7beta1 integrin mediates a response to tenascin-C and the first to demonstrate a functional role for the alpha7beta1 integrin receptor in CNS neurons.

Bone marrow stromal cells stimulate neurite outgrowth over neural proteoglycans (CSPG), myelin associated glycoprotein and Nogo-A

In animal models, transplantation of bone marrow stromal cells (MSC) into the spinal cord following injury enhances axonal regeneration and promotes functional recovery. How these improvements come about is currently unclear. We have examined the interaction of MSC with neurons, using an established in vitro model of nerve growth, in the presence of substrate-bound extracellular molecules that are thought to inhibit axonal regeneration, i.e., neural proteoglycans (CSPG), myelin associated glycoprotein (MAG) and Nogo-A. Each of these molecules repelled neurite outgrowth from dorsal root ganglia (DRG) in a concentration-dependent manner. However, these nerve-inhibitory effects were much reduced in MSC/DRG co-cultures. Video microscopy demonstrated that MSC acted as "cellular bridges" and also "towed" neurites over the nerve-inhibitory substrates. Whereas conditioned medium from MSC cultures stimulated DRG neurite outgrowth over type I collagen, it did not promote outgrowth over CSPG, MAG or Nogo-A. These findings suggest that MSC transplantation may promote axonal regeneration both by stimulating nerve growth via secreted factors and also by reducing the nerve-inhibitory effects of the extracellular molecules present.

Spinal motor neurite outgrowth over glial scar inhibitors is enhanced by coculture with bone marrow stromal cells

Background context Transplantation of bone marrow cells into spinal cord lesions promotes functional recovery in animal models, and recent clinical trials suggest possible recovery also in humans. The mechanisms responsible for these improvements are still unclear. Purpose To characterize spinal cord motor neurite interactions with human bone marrow stromal cells (MSCs) in an in vitro model of spinal cord injury (SCI). Study design/setting Previously, we have reported that human MSCs promote the growth of extending sensory neurites from dorsal root ganglia (DRG), in the presence of some of the molecules present in the glial scar, which are attributed with inhibiting axonal regeneration after SCI. We have adapted and optimized this system replacing the DRG with a spinal cord culture to produce a central nervous system (CNS) model, which is more relevant to the SCI situation. Methods We have developed and characterized a novel spinal cord culture system. Human MSCs were cocultured with spinal motor neurites in substrate choice assays containing glial scar-associated inhibitors of nerve growth. In separate experiments, MSC-conditioned media were analyzed and added to spinal motor neurites in substrate choice assays. Results As has been reported previously with DRG, substrate-bound neurocan and Nogo-A repelled spinal neuronal adhesion and neurite outgrowth, but these inhibitory effects were abrogated in MSC/spinal cord cocultures. However, unlike DRG, spinal neuronal bodies and neurites showed no inhibition to substrates of myelin-associated glycoprotein. In addition, the MSC secretome contained numerous neurotrophic factors that stimulated spinal neurite outgrowth, but these were not sufficient stimuli to promote spinal neurite extension over inhibitory concentrations of neurocan or Nogo-A. Conclusions These findings provide novel insight into how MSC transplantation may promote regeneration and functional recovery in animal models of SCI and in the clinic, especially in the chronic situation in which glial scars (and associated neural inhibitors) are well established. In addition, we have confirmed that this CNS model predominantly comprises motor neurons via immunocytochemical characterization. We hope that this model may be used in future research to test various other potential interventions for spinal injury or disease states. © 2014 Elsevier Inc. All rights reserved.

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems. © 2010 Elsevier Inc.

A small molecule activator of p300/CBP histone acetyltransferase promotes survival and neurite growth in a cellular model of Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterised by motor and non-motor symptoms, resulting from the degeneration of nigrostriatal dopaminergic neurons and peripheral autonomic neurons. Given the limited success of neurotrophic factors in clinical trials, there is a need to identify new small molecule drugs and drug targets to develop novel therapeutic strategies to protect all neurons that degenerate in PD. Epigenetic dysregulation has been implicated in neurodegenerative disorders, while targeting histone acetylation is a promising therapeutic avenue for PD. We and others have demonstrated that histone deacetylase inhibitors have neurotrophic effects in experimental models of PD. Activators of histone acetyltransferases (HAT) provide an alternative approach for the selective activation of gene expression, however little is known about the potential of HAT activators as drug therapies for PD. To explore this potential, the present study investigated the neurotrophic effects of CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide), which is a potent small molecule activator of the histone acetyltransferase p300/CBP, in the SH-SY5Y neuronal cell line. We report that CTPB promoted the survival and neurite growth of the SH-SY5Y cells, and also protected these cells from cell death induced by the neurotoxin 6-hydroxydopamine. This study is the first to investigate the phenotypic effects of the HAT activator CTPB, and to demonstrate that p300/CBP HAT activation has neurotrophic effects in a cellular model of PD.

Ocular masquerade syndrome associated with extranodal nasal natural killer/T-cell lymphoma: case report

A 33-year-old woman complained of unilateral eyelid edema and blurred vision. Initial ophthalmic examination disclosed anterior chamber reaction with keratic precipitates on the cornea, without posterior abnormalities. Anterior uveitis was treated. Despite that, patient showed rapidly progressive unilateral vision loss with optic nerve swelling. Systemic workup was inconclusive, as well as cranial magnetic resonance imaging and cerebrospinal fluid examination. Based on the hypothesis of optic neuritis, intravenous methylprednisolone pulse was performed with no success. During the following days, the patient presented pericardial effusion and cardiac tamponade, progressing to death. Necropsy was performed and diagnosis of extranodal natural killers/T-cell lymphoma, nasal type with ocular involvement was confirmed by immunohistochemistry.

Expression of galectin-1 in the mouse olfactory system

Primary sensory olfactory axons arise from the olfactory neuroepithelium that lines the nasal cavity and then project via the olfactory nerve into the olfactory bulb. The P-galactoside binding lectin, galectin-1,and its laminin ligand have been implicated in the growth of these axons along this pathway. In galectin-1 null mutant mice, a subpopulation of primary sensory olfactory axons fails to reach its targets in the olfactory bulb. In the present study we examined the spatiotemporal expression pattern of galectin-1 in normal mice in order to understand its role in the development of the olfactory nerve pathway. At E15.5, when olfactory axons have already contacted the olfactory bulb, galectin-1 was expressed in the cartilage and mesenchyme surrounding the nasal cavity but was absent from the olfactory neuroepithelium, nerve and bulb. Between E16.5 and birth galectin-1 began to be expressed by olfactory nerve ensheathing cells in the lamina propria of the neuroepithelium and nerve fibre layer. Galectin-1 was neither expressed by primary sensory neurons in the olfactory neuroepithelium nor by their axons in the olfactory nerve. Laminin, a galectin-1 ligand, also exhibited a similar expression pattern in the embryonic olfactory nerve pathway. Our results reveal that galectin-1 is dynamically expressed by glial elements within the nerve fibre layer during a discrete period in the developing olfactory nerve pathway. Previous studies have reported galectin-1 acts as a substrate adhesion molecule by cross-linking primary sensory olfactory neurons to laminin. Thus, the coordinate expression of galectin-1 and laminin in the embryonic nerve fibre layer suggests that these molecules support the adhesion and fasciculation of axons en route to their glomerular targets.

Chondroitin sulfates modulate axon guidance in embryonic Xenopus brain

Chondroitin sulfate proteoglycans display both inhibitory and stimulatory effects on cell adhesion and neurite outgrowth in vitro. The functional activity of these proteoglycans appears to be context specific and dependent on the presence of different chondroitin sulfate-binding molecules. Little is known about the role of chondroitin sulfate proteoglycans in the growth and guidance of axons in vivo. To address this question, we examined the effects of exogenous soluble chondroitin sulfates on the growth and guidance of axons arising from a subpopulation of neurons in the vertebrate brain which express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM. Intact brains of stage 28 Xenopus embryos were unilaterally exposed to medium containing soluble exogenous chondroitin sulfates. When exposed to chondroitin sulfate, NOC-2(+) axons within the tract of the postoptic commissure failed to follow their normal trajectory across the ventral midline via the ventral commissure in the midbrain. Instead, these axons either stalled or grew into the dorsal midbrain or continued growing longitudinally within the ventral longitudinal tract. These findings suggest that chondroitin sulfate proteoglycans indirectly modulate the growth and guidance of a subpopulation of forebrain axons by regulating either matrix-bound or cell surface cues at specific choice points within the developing vertebrate brain. (C) 1998 Academic Press.

Novel guidance cues during neuronal pathfinding in the early scaffold of axon tracts in the rostral brain

A scaffold of axons consisting of a pair of longitudinal tracts and several commissures is established during early development of the vertebrate brain. We report here that NOC-2, a cell surface carbohydrate, is selectively expressed by a subpopulation of growing axons in this scaffold in Xenopus. NOC-2 is present on two glycoproteins, one of which is a novel glycoform of the neural cell adhesion molecule N-CAM. When the function of NOC-2 was perturbed using either soluble carbohydrates or anti-NOC-2 antibodies, axons expressing NOC-2 exhibited aberrant growth at specific points in their pathway. NOC-2 is the first-identified axon guidance molecule essential for development of the axon scaffold in the embryonic vertebrate brain.

Structure/function studies of S100A8/A9

The functional importance of members of the S100 Ca2+-binding protein family is recently emerging. A variety of activities, several of which are apparently opposing, are attributed to S100A8, a protein implicated in embryogenesis, growth, differentiation, and immune and inflammatory processes. Murine (m) S100A8 was initially described as a chemoattractant (CP-10) for myeloid cells. It is coordinately expressed with mS100A9 (MRP14) in neutrophils and the non-covalent heterodimer is presumed to be the functional intracellular species. The extracellular chemotactic activity of mS100A8, however, is not dependent on mS100A9 and occurs at concentrations (10(-13)-10(-11) M) at which the non-covalent heterodimer would probably dissociate. This review focuses on the structure and post-translational modifications of mS100A8/A9 and their effects on function, particularly chemotaxis.