Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons

Development of appropriate dendritic arbors is crucial for neuronal information transfer. We show, using seizure-related gene 6 (sez-6) null mutant mice, that Sez-6 is required for normal dendritic arborization of cortical neurons. Deep-layer pyramidal neurons in the somatosensory cortex of sez-6 null mice exhibit an excess of short dendrites, and cultured cortical neurons lacking Sez-6 display excessive neurite branching. Overexpression of individual Sez-6 isoforms in knockout neurons reveals opposing actions of membrane-bound and secreted Sez-6 proteins, with membrane-bound Sez-6 exerting an antibranching effect under both basal and depolarizing conditions. Layer V pyramidal neurons in knockout brain slices show reduced excitatory postsynaptic responses and a reduced dendritic spine density, reflected by diminished punctate staining for postsynaptic density 95 (PSD-95). In behavioral tests, the sez-6 null mice display specific exploratory, motor, and cognitive deficits. In conclusion, cell-surface protein complexes involving Sez-6 help to sculpt the dendritic arbor, in turn enhancing synaptic connectivity.     

Sez-6 may play an important role in neurite outgrowth through the PKCgamma signaling pathways

Seizure-related gene 6 (sez-6) was originally identified in a study of pentylenetetrazole-treated cortical neurons. Further studies on the structure and expression pattern suggested that Sez-6 may play an important role in neuronal development and function. In the present study, PC12 cells were used as a model to investigate the role of Sez-6 in neurite outgrowth. After a period of NGF treatment, the expression of Sez-6 in PC12 cells was increased. When Sez-6 expression was suppressed by the addition of an effective short hairpin RNA (shRNA) plasmid, the neurite outgrowth was significantly inhibited. In addition, we detected the expression level of protein kinase Cgamma (PKCgamma), and found that the PKCgamma protein level was reduced in the differentiated PC12 cells but increased in PC12 cells lacking Sez-6. Taken together, our results indicate that Sez-6 acts on the neurite outgrowth of PC12 cells likely through the PKCgamma signaling pathways.     

Seizure-Related Gene 6 (Sez-6) in Amacrine Cells of the Rodent Retina and the Consequence of Gene Deletion

Background

Seizure-related gene 6 (Sez-6) is expressed in neurons of the mouse brain, retina and spinal cord. In the cortex, Sez-6 plays a role in specifying dendritic branching patterns and excitatory synapse numbers during development.

Methodology/Principal Findings

The distribution pattern of Sez-6 in the retina was studied using a polyclonal antibody that detects the multiple isoforms of Sez-6. Prominent immunostaining was detected in GABAergic, but not in AII glycinergic, amacrine cell subpopulations of the rat and mouse retina. Amacrine cell somata displayed a distinct staining pattern with the Sez-6 antibody: a discrete, often roughly triangular-shaped bright spot positioned between the nucleus and the apical dendrite superimposed over weaker general cytoplasmic staining. Displaced amacrines in the ganglion cell layer were also positive for Sez-6 and weaker staining was occasionally observed in neurons with the morphology of alpha ganglion cells. Two distinct Sez-6 positive strata were present in the inner plexiform layer in addition to generalized punctate staining. Certain inner nuclear layer cells, including bipolar cells, stained more weakly and diffusely than amacrine cells, although some bipolar cells exhibited a perinuclear “bright spot” similar to amacrine cells. In order to assess the role of Sez-6 in the retina, we analyzed the morphology of the Sez-6 knockout mouse retina with immunohistochemical markers and compared ganglion cell dendritic arbor patterning in Sez-6 null retinae with controls. The functional importance of Sez-6 was assessed by dark-adapted paired-flash electroretinography (ERG).

Conclusions

In summary, we have reported the detailed expression pattern of a novel retinal marker with broad cell specificity, useful for retinal characterization in rodent experimental models. Retinal morphology, ganglion cell dendritic branching and ERG waveforms appeared normal in the Sez-6 knockout mouse suggesting that, in spite of widespread expression of Sez-6, retinal function in the absence of Sez-6 is not affected.     

NeuronGrowth, a software for automatic quantification of neurite and filopodial dynamics from time-lapse sequences of digital images

We developed NeuronGrowth, a software for the automatic quantification of extension and retraction of neurites and filopodia, from time-lapse sequences of two-dimensional digital micrographs. NeuronGrowth requires a semiautomatic characterization of individual neurites in a reference frame, which is then used for automatic tracking and measurement of every neurite over the whole image sequence. Modules for sequence alignment, background subtraction, flat field correction, light normalization, and cropping have been integrated to improve the quality of the analysis. Moreover, NeuronGrowth incorporates a deconvolution filter that corrects the shadow-cast effect of differential interference contrast (DIC) images. NeuronGrowth was tested by analyzing the formation of outgrowth patterns by individual leech neurons cultured under two different conditions. Phase contrast images were obtained from neurons plated on CNS homogenates and DIC images were obtained from similar neurons plated on ganglion capsules as substrates. Filopodia were measured from fluorescent growth-cones of chick dorsal root ganglion cells. Quantitative data of neurite extension and retraction obtained by three different users applying NeuronGrowth and two other manually operated software packages were similar. However, NeuronGrowth required less user participation and had a better time performance when compared with the other software packages. NeuronGrowth may be used in general to quantify the dynamics of tubular structures such as blood vessels. NeuronGrowth is a free plug-in for the free software ImageJ and can be downloaded along with a user manual, a troubleshooting section and other information required for its use from http://www.ifc.unam.mx or http://www.ifc.unam.mx/ffm/index.html.     

Automated screening of neurite outgrowth

Outgrowth of neurites in culture is used for assessing neurotrophic activity. Neurite measurements have been performed very slowly using manual methods or more efficiently with interactive image analysis systems. In contrast, medium-throughput and noninteractive image analysis of neurite screens has not been well described. The authors report the performance of an automated image acquisition and analysis system (IN Cell Analyzer 1000) in the neurite assay. Neuro-2a (N2a) cells were plated in 96-well plates and were exposed to 6 conditions of retinoic acid. Immunofluorescence labeling of the cytoskeleton was used to detect neurites and cell bodies. Acquisition of the images was automatic. The image set was then analyzed by both manual tracing and automated algorithms. On 5 relevant parameters (number of neurites, neurite length, total cell area, number of cells, neurite length per cell), the authors did not observe a difference between the automated analysis and the manual analysis done by tracing. These data suggest that the automated system addresses the same biology as human scorers and with the same measurement precision for treatment effects. However, throughput of the automated system is orders of magnitude higher than with manual methods.     

Docosahexaenoic acid promotes neurite growth in hippocampal neurons

Docosahexanoic acid (22:6n-3; DHA) deficiency during development is associated with impairment in learning and memory, suggesting an important role of DHA in neuronal development. Here we provide evidence that DHA promotes neuronal differentiation in rat embryonic hippocampal primary cultures. DHA deficiency in vitro was spontaneously induced by culturing hippocampal cells in chemically defined medium. DHA supplementation improved DHA levels to values observed in freshly isolated hippocampus. We found that DHA supplementation in culture increased the population of neurons with longer neurite length per neuron and with higher number of branches. However, supplementation with arachidonic, oleic or docosapentaenoic acid did not have any effect, indicating specificity of the DHA action on neurite growth. Furthermore, hippocampal cultures obtained from n-3 fatty acid deficient animals contained a lower DHA level and a neuronal population with shorter neurite length per neuron in comparison to those obtained from animals with adequate n-3 fatty acids. DHA supplementation to the deficient group recovered the neurite length to the level similar to n-3 fatty acid adequate cultures. Our data demonstrates that DHA uniquely promotes neurite growth in hippocampal neurons. Inadequate neurite development due to DHA deficiency may contribute to the cognitive impairment associated with n-3 fatty acid deficiency.     

3D multicolor super-resolution imaging offers improved accuracy in neuron tracing

The connectivity among neurons holds the key to understanding brain function. Mapping neural connectivity in brain circuits requires imaging techniques with high spatial resolution to facilitate neuron tracing and high molecular specificity to mark different cellular and molecular populations. Here, we tested a three-dimensional (3D), multicolor super-resolution imaging method, stochastic optical reconstruction microscopy (STORM), for tracing neural connectivity using cultured hippocampal neurons obtained from wild-type neonatal rat embryos as a model system. Using a membrane specific labeling approach that improves labeling density compared to cytoplasmic labeling, we imaged neural processes at 44 nm 2D and 116 nm 3D resolution as determined by considering both the localization precision of the fluorescent probes and the Nyquist criterion based on label density. Comparison with confocal images showed that, with the currently achieved resolution, we could distinguish and trace substantially more neuronal processes in the super-resolution images. The accuracy of tracing was further improved by using multicolor super-resolution imaging. The resolution obtained here was largely limited by the label density and not by the localization precision of the fluorescent probes. Therefore, higher image resolution, and thus higher tracing accuracy, can in principle be achieved by further improving the label density.     

tPA Regulates Neurite Outgrowth by Phosphorylation of LRP5/6 in Neural Progenitor Cells

Despite the important role of tissue plasminogen activator (tPA) as a neuromodulator in neurons, microglia, and astrocytes, its role in neural progenitor cell (NPC) development is not clear yet. We identified that tPA is highly expressed in NPCs compared with neurons. Inhibition of tPA activity or expression using tPA stop, PAI-1, or tPA siRNA inhibited neurite outgrowth from NPCs, while overexpression or addition of exogenous tPA increased neurite outgrowth. The expression of Wnt and β-catenin as well as phosphorylation of LRP5 and LRP6, which has been implicated in Wnt–β-catenin signaling, was rapidly increased after tPA treatment and was decreased by tPA siRNA transfection. Knockdown of β-catenin or LRP5/6 expression by siRNA prevented tPA-induced neurite extension. NPCs obtained from tPA KO mice showed impaired neurite outgrowth compared with WT NPCs. In ischemic rat brains, axon density was higher in the brains transplanted with WT NPCs than in those with tPA KO NPCs, suggesting increased axonal sprouting by NPC-derived tPA. tPA-mediated regulation of neuronal maturation in NPCs may play an important role during development and in regenerative conditions.     

A Simple and Reliable Technique to Combine Oligonucleotide Probe in Situ Hybridization with Neuronal Tract Tracing in Vertebrate Embryos

We describe a simple and reliable combination of in situ hybridization with neuronal tracing. The technique uses recent advances in the field of neuronal tract tracing including fast diffusing, low molecular weight dextran amines and fade resistant fluorescent dyes, and combines them with in situ hybridization using a sensitive Oligonucleotide probe. Using this technique we have investigated the mRNA encoding the trkB receptor for brain-derived neurotrophic factor in identified facial and vestibular afferent and efferent neurons. We found very low levels of trkB mRNA in facial efferent neurons, whereas in the vestibular afferent neurons, clear labeling for the trkB mRNA could be seen. This technique can be applied to the developing embryo to study topology of a variety of cellular markers with reference to neuronal population or fibers identified by their origin or target.     

Rapid Reconstruction of 3D Neuronal Morphology from Light Microscopy Images with Augmented Rayburst Sampling

Digital reconstruction of three-dimensional (3D) neuronal morphology from light microscopy images provides a powerful technique for analysis of neural circuits. It is time-consuming to manually perform this process. Thus, efficient computer-assisted approaches are preferable. In this paper, we present an innovative method for the tracing and reconstruction of 3D neuronal morphology from light microscopy images. The method uses a prediction and refinement strategy that is based on exploration of local neuron structural features. We extended the rayburst sampling algorithm to a marching fashion, which starts from a single or a few seed points and marches recursively forward along neurite branches to trace and reconstruct the whole tree-like structure. A local radius-related but size-independent hemispherical sampling was used to predict the neurite centerline and detect branches. Iterative rayburst sampling was performed in the orthogonal plane, to refine the centerline location and to estimate the local radius. We implemented the method in a cooperative 3D interactive visualization-assisted system named flNeuronTool. The source code in C++ and the binaries are freely available at http://sourceforge.net/projects/flneurontool/. We validated and evaluated the proposed method using synthetic data and real datasets from the Digital Reconstruction of Axonal and Dendritic Morphology (DIADEM) challenge. Then, flNeuronTool was applied to mouse brain images acquired with the Micro-Optical Sectioning Tomography (MOST) system, to reconstruct single neurons and local neural circuits. The results showed that the system achieves a reasonable balance between fast speed and acceptable accuracy, which is promising for interactive applications in neuronal image analysis.     

Developmental regulation of plasticity along neurite shafts

Although it is becoming increasingly clear that structural dynamics on neurite shafts play important roles in establishing neuronal architecture, the underlying mechanisms are unknown. The present study investigates local induction of filopodia along the shafts of neurites, a process that, by analogy to the growth cone, can represent the first stage in the generation of a new neuronal process. We show that filopodia can be induced reliably along the neurite shaft in response to a localized electric field stimulus that evokes large local intracellular calcium increases. Neither induction of filopodia nor a local rise in intracellular calcium occurred in calcium free medium. Although calcium induction of neurite filopodia is highly reliable, forming in response to more than 90% of attempts, it is developmental state-dependent, since neurite filopodia could not be induced in neurons previously defined as “stable state.” We have found two distinct changes in stable state neurons that can decrease the ability to induce new neurites. The first is a reduced calcium response: Field stimulation produced large local rises (280 nM) in stable state neurons. Second, stable state neurons change so that even when the stimulus intensity was increased to elicit a calcium response that would have been sufficient to induce filopodia in growing neurites, neurite filopodia were still not induced. Thus, intracellular calcium plays a key role in structural changes along the shafts of neurites. Furthermore, developmental changes in both calcium homeostatic components, and in calcium responsiveness (i.e., the sensitivity of cellular components that modulate neurite morphology) underlie shifts from plasticity to stability of neuronal architecture in this system. © 1995 John Wiley & Sons, Inc.     

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.     

A novel method for simultaneous anterograde and retrograde labeling of spinal cord motor tracts in the same animal.

Examination of repaired spinal cord tracts has usually required separate groups of animals for anterograde and retrograde tracing owing to the incompatibility of techniques such as tissue fixation. However, anterograde and retrograde labeling of different animals subjected to the same repair may not allow accurate examination of that repair strategy because widely variable results can occur in animals subjected to the same strategy. We have developed a reliable method of labeling spinal cord motor tracts bidirectionally in the same animal using DiI, a lipophilic dye, to anterogradely label the corticospinal tract and Fluoro-Gold (FG) to retrogradely label cortical and brainstem neurons of several spinal cord motor tracts in normal and injured adult rats. Other tracer combinations (lipophilic dyes or fluorescent dextrans) were also investigated but were less effective. We also developed methods to minimize autofluorescence with the DiI/FG technique, and found that the DiI/FG technique is compatible with decalcification and immunohistochemistry for several markers relevant for studies of spinal cord regeneration. Thus, the use of anterograde DiI and retrograde FG is a novel technique for bidirectional labeling of the motor tracts of the adult spinal cord with fluorescent tracers and should be useful for demonstrating neurite regeneration in studies of spinal cord repair.(J Histochem Cytochem 49:1111-1122, 2001)     

Identification of small molecule inhibitors of neurite loss induced by Aβ peptide using high content screening

Multiple lines of evidence indicate a strong relationship between Αβ peptide-induced neurite degeneration and the progressive loss of cognitive functions in Alzheimer disease (AD) patients and in AD animal models. This prompted us to develop a high content screening assay (HCS) and Neurite Image Quantitator (NeuriteIQ) software to quantify the loss of neuronal projections induced by Aβ peptide neurons and enable us to identify new classes of neurite-protective small molecules, which may represent new leads for AD drug discovery. We identified thirty-six inhibitors of Aβ-induced neurite loss in the 1,040-compound National Institute of Neurological Disorders and Stroke (NINDS) custom collection of known bioactives and FDA approved drugs. Activity clustering showed that non-steroidal anti-inflammatory drugs (NSAIDs) were significantly enriched among the hits. Notably, NSAIDs have previously attracted significant attention as potential drugs for AD; however their mechanism of action remains controversial. Our data revealed that cyclooxygenase-2 (COX-2) expression was increased following Aβ treatment. Furthermore, multiple distinct classes of COX inhibitors efficiently blocked neurite loss in primary neurons, suggesting that increased COX activity contributes to Aβ peptide-induced neurite loss. Finally, we discovered that the detrimental effect of COX activity on neurite integrity may be mediated through the inhibition of peroxisome proliferator-activated receptor γ (PPARγ) activity. Overall, our work establishes the feasibility of identifying small molecule inhibitors of Aβ-induced neurite loss using the NeuriteIQ pipeline and provides novel insights into the mechanisms of neuroprotection by NSAIDs.     

An Isogeometric Analysis Computational Platform for Material Transport Simulation in Complex Neurite Networks

Neurons exhibit remarkably complex geometry in their neurite networks. So far, how materials are transported in the complex geometry for survival and function of neurons remains an unanswered question. Answering this question is fundamental to understanding the physiology and disease of neurons. Here, we have developed an isogeometric analysis (IGA) based platform for material transport simulation in neurite networks. We modeled the transport process by reaction-diffusion-transport equations and represented geometry of the networks using truncated hierarchical tricubic B-splines (THB-spline3D). We solved the Navier-Stokes equations to obtain the velocity field of material transport in the networks. We then solved the transport equations using the streamline upwind/Petrov-Galerkin (SU/PG) method. Using our IGA solver, we simulated material transport in three basic models of the network geometry: a single neurite, a neurite bifurcation, and a neurite tree with three bifurcations. In addition, the robustness of our solver is illustrated by simulating material transport in three representative and complex neurite networks. From the simulation we discovered several spatial patterns of the transport process. Together, our simulation provides key insights into how material transport in neurite networks is mediated by their complex geometry.     

Laminin promotes neuritic regeneration from cultured peripheral and central neurons

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co- purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.     

Effect of Na(+), K(+)-pump inhibitors on the sensory ganglia neurite growth

Inhibitors of Na(+), K(+)-pump belonging to the class of cardiac glycosides were investigated in organotypic tissue culture of dorsal root ganglia cells of 10-12 days old chicken embryos. The data obtained show that the application of cardiac glycosides (strophantin K and digoxin) in a wide range of concentrations controls the neurite growth in sensory neurons in the dose-dependent manner. It was shown, that at the concentrations of cardiac glycoside exceeding 1 x 10(-6) M the growth of neurites was totally inhibited. Our data indicate that cardiac glycoside have the down-regulation effect on the neurite growth. The data obtained indicate that the Na(+), K(+)-ATPase is involved in the control of the process of neurite growth as a signal transducer.     

Neurite outgrowth from progeny of epidermal growth factor-responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: effect of brain-derived neurotrophic factor

Epidermal growth factor (EGF)-responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self-renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF-responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF-generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain-derived neurotrophic factor (BDNF) (5 ng in 0.5 microL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF-responsive stem cell-derived neurons possess limited intrinsic capability for long-distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF-responsive stem cell-derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF.     

Directional control of neurite outgrowth from cultured hippocampal neurons is modulated by the lectin concanavalin A.

Cell surface carbohydrates play an important role in the regulation of neurite outgrowth during neuronal development. We have investigated the actions of the plant lectin concanavalin A (Con A), a carbohydrate-binding protein, on neurite outgrowth from hippocampal pyramidal neurons in primary cell culture. Neurons plated in culture medium containing nanomolar concentrations of Con A have a larger number of primary neurites arising directly from the cell soma than do neurons plated in culture medium alone. Furthermore, Con A causes counterclockwise turning of neurites in over 70% of the cultured neurons. Both of these effects of Con A are blocked by the hapten sugar alpha-methyl-D-mannopyranoside, suggesting that they result from the interaction of Con A with a cell surface carbohydrate. Another lectin with a different sugar specificity, wheat germ agglutinin, does not modulate neurite outgrowth. Analysis of neurite outgrowth using video-enhanced microscopy reveals that the counterclockwise turning is accompanied by directionally biased extension of filopodia from the growth cones of growing neurites. Treatment of the neurons with cytochalasin, which disrupts actin polymerization, eliminates the neurite turning induced by Con A, suggesting that actin microfilaments are involved in directional control of neurite outgrowth.     

Th1 cells promote neurite outgrowth from cortical neurons via a mechanism dependent on semaphorins

The roles of T lymphocytes in the central nervous system (CNS) are diverse; their roles in the injured CNS have been reported to be both detrimental and advantageous. Hence, an investigation of the effects of specific subsets of T cells on neurons may provide an insight into the interaction between the nervous system and the immune system. In the present study, we demonstrate that a specific subset of T lymphocytes enhanced neurite outgrowth in vitro. When cultured T helper type 1 (Th1) cells were co-cultured with cortical neurons, neurite outgrowth from neurons was enhanced; however, the same was not observed when Th2 or naïve T cells were used. We observed that the promotion of neurite outgrowth by Th1 cells was completely inhibited by anti-interferon γ (IFN-γ) neutralizing antibody, but that IFN-γ did not directly promote neurite growth. Furthermore, experiments using knockout mice revealed that semaphorin 4A (Sema4A) but not Sema7A was required for the effect produced by Th1 cells. These results demonstrate that Sema4A and IFN-γ expressed in Th1 cells play a critical role in enhancing neurite outgrowth from cortical neurons.