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Neurons of the central nervous system (CNS) that project long axons into the spinal cord have a poor axon regenerative capacity compared to neurons of the peripheral nervous system. The corticospinal tract (CST) is particularly notorious for its poor regeneration. Because of this, traumatic spinal cord injury (SCI) is a devastating condition that remains as yet uncured. Based on our recent observations that direct neuronal interleukin-4 (IL-4) signaling leads to repair of axonal swellings and beneficial effects in neuroinflammation, we hypothesized that IL-4 acts directly on the CST. Here, we developed a tissue culture model for CST regeneration and found that IL-4 promoted new growth cone formation after axon transection. Most importantly, IL-4 directly increased the regenerative capacity of both murine and human CST axons, which corroborates its regenerative effects in CNS damage. Overall, these findings serve as proof-of-concept that our CST regeneration model is suitable for fast screening of new treatments for SCI.

Altered synaptic bioactive lipid signaling has been recently shown to augment neuronal excitation in the hippocampus of adult animals by activation of presynaptic LPA2-receptors leading to increased presynaptic glutamate release. Here, we show that this results in higher postsynaptic Ca2+ levels and in premature onset of spontaneous neuronal activity in the developing entorhinal cortex. Interestingly, increased synchronized neuronal activity led to reduced axon growth velocity of entorhinal neurons which project via the perforant path to the hippocampus. This was due to Ca2+-dependent molecular signaling to the axon affecting stabilization of the actin cytoskeleton. The spontaneous activity affected the entire entorhinal cortical network and thus led to reduced overall axon fiber numbers in the mature perforant path that is known to be important for specific memory functions. Our data show that precise regulation of early cortical activity by bioactive lipids is of critical importance for proper circuit formation., (© The Author 2016. Published by Oxford University Press.)

A body of experimental evidence suggests that T cells mediate neuroprotection following CNS injury; however, the antigen specificity of these T cells and how they mediate neuroprotection are unknown. Here, we have provided evidence that T cell-mediated neuroprotection after CNS injury can occur independently of major histocompatibility class II (MHCII) signaling to T cell receptors (TCRs). Using two murine models of CNS injury, we determined that damage-associated molecular mediators that originate from injured CNS tissue induce a population of neuroprotective, IL-4-producing T cells in an antigen-independent fashion. Compared with wild-type mice, IL-4-deficient animals had decreased functional recovery following CNS injury; however, transfer of CD4+ T cells from wild-type mice, but not from IL-4-deficient mice, enhanced neuronal survival. Using a culture-based system, we determined that T cell-derived IL-4 protects and induces recovery of injured neurons by activation of neuronal IL-4 receptors, which potentiated neurotrophin signaling via the AKT and MAPK pathways. Together, these findings demonstrate that damage-associated molecules from the injured CNS induce a neuroprotective T cell response that is independent of MHCII/TCR interactions and is MyD88 dependent. Moreover, our results indicate that IL-4 mediates neuroprotection and recovery of the injured CNS and suggest that strategies to enhance IL-4-producing CD4+ T cells have potential to attenuate axonal damage in the course of CNS injury in trauma, inflammation, or neurodegeneration.

Precise crosstalk between the nervous and immune systems is important for neuroprotection and axon plasticity after injury. Recently, we demonstrated that IL-1β acts as a potent inducer of neurite outgrowth from organotypic brain slices in vitro, suggesting a potential function of IL-1β in axonal plasticity. Here, we have investigated the effects of IL-1β on axon plasticity during glial scar formation and on functional recovery in a mouse model of spinal cord compression injury (SCI). We used an IL-1β deficiency model (IL-1βKO mice) and administered recombinant IL-1β. In contrast to our hypothesis, the histological analysis revealed a significantly increased lesion width and a reduced number of corticospinal tract fibers caudal to the lesion center after local application of recombinant IL-1β. Consistently, the treatment significantly worsened the neurological outcome after SCI in mice compared with PBS controls. In contrast, the absence of IL-1β in IL-1βKO mice significantly improved recovery from SCI compared with wildtype mice. Histological analysis revealed a smaller lesion size, reduced lesion width and greatly decreased astrogliosis in the white matter, while the number of corticospinal tract fibers increased significantly 5 mm caudal to the lesion in IL-1βKO mice relative to controls. Our study for the first time characterizes the detrimental effects of IL-1β not only on lesion development (in terms of size and glia activation), but also on the plasticity of central nervous system axons after injury.

In this study, we investigated the hypothesis whether neurotrophins have a differential influence on neurite growth from the entorhinal cortex depending on the presence or absence of hippocampal target tissue. We investigated organotypic brain slices derived from the entorhinal-hippocampal system to analyze the effects of endogenous and recombinant neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) on neurite outgrowth and reinnervation. In the reinnervation assay, entorhinal cortex explants of transgenic mice expressing enhanced green fluorescent protein (EGFP) were co-cultured with wild-type hippocampi under the influence of recombinant NT-3 and NT-4 (500 ng/ml). Both recombinant NT-3 and NT-4 significantly increased the growth of EGFP+ nerve fibers into the target tissue. Consistently, reinnervation of the hippocampi of NT-4(-/-) and NT-3(+/-)NT-4(-/-) mice was substantially reduced. In contrast, the outgrowth assay did not exhibit reduction in axon outgrowth of NT-4(-/-) or NT-3(+/-)NT-4(-/-) cortex explants, while the application of recombinant NT-3 (500 ng/ml) induced a significant increase in the neurite extension of cortex explants. Recombinant NT-4 had no effect. In summary, only recombinant NT-3 stimulates axon outgrowth from cortex explants, while both endogenous and recombinant NT-3 and NT-4 synergistically promote reinnervation of the denervated hippocampus. These results suggest that endogenous and exogenous NT-3 and NT-4 differentially influence neurite growth depending on the presence or absence of target tissue.

Recently it has become clear that the neuronal compartment plays a more important role than previously thought in the pathology of multiple sclerosis. Apart from demyelination, neuronal pathology is apparently largely responsible for the brain atrophy that can be observed early on and throughout the course of the disease. The loss of axons and their neurons in the course of chronic neuroinflammation is a major factor determining long-term disability in patients. The actual steps leading from immune attack against the myelin sheath to neuronal damage are not yet fully clear. Here we review key findings about direct axonal damage processes, demyelination-related neuronal pathology and cell-body pathology, the major pathologic correlates that underlie brain atrophy in MS., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Members of the plasticity-related gene (PRG1-4) family are brain-specific integral membrane proteins and implicated in neuronal plasticity, such as filopodia formation and axon growth after brain lesion. Here we report on the cloning of a novel member of the PRG family, PRG5, with high homologies to PRG3. PRG5 is regulated during brain and spinal cord development and is exclusively allocated within the nervous system. When introduced in neurons, PRG5 is distributed in the plasma membrane and induces filopodia as well as axon elongation and growth. Conversely, siRNA mediated knockdown of PRG5 impedes axon growth and disturbs filopodia formation. Here we show that PRG5 induces filopodia growth independently of Cdc42. Moreover, axon collapse and RhoA activation induced by LPA and myelin-associated neurite inhibitor Nogo-A is attenuated in the presence of PRG5, although direct activation of the RhoA-Rho-PIP5K kinase pathway abolishes PRG5 -formed neurites. Thus, we describe here the identification of a novel member of the PRG family that induces filopodia and axon elongation in a Cdc42-independent manner. In addition, PRG5 impedes brain injury-associated growth inhibitory signals upstream of the RhoA-Rho kinase pathway.

Inflammation is part of the physiological wound healing response following mechanical lesioning of the peripheral nervous system. However, cytokine effects on axonal regeneration are still poorly understood. Because cytokines influence the expression of neurotrophins and their receptors, which play a major role in axonal outgrowth after lesioning, we investigated the hypothesis that cytokines influence specifically neurotrophin-dependent axon elongation. Therefore, we have characterized neurotrophin-dependent neurite outgrowth of murine dorsal root ganglia (DRG) in vitro and investigated the influence of pro- and anti-inflammatory cytokines on these outgrowth patterns. Embryonic day 13 (E13) DRG were cultured in Matrigel for 2 days and axonal morphology, density and elongation were determined using an image analysis system. Nerve growth factor (NGF), neurotrophin-3 (NT-3) and -4 (NT-4) were applied alone (50 ng/mL), in double or in triple combinations. NT-3, NT-4 and NT-3 + NT-4 combined induced a moderate increase in axonal outgrowth (P < 0.001) compared with controls, while NGF and all combinations including NGF induced an even more pronounced increase in axonal outgrowth (P < 0.001). After characterizing these outgrowth patterns, interleukin (IL)-1beta, IL-4, IL-6, interferon-gamma (IFNgamma) and tumour necrosis factor-alpha (TNFalpha) (50 or 500 ng/mL) were added to the different neurotrophin combinations. Low doses of TNFalpha and IL-6 influenced neurite extension induced by endogenous neurotrophins. IL-4 increased NT-4-induced outgrowth. IL-6 stimulated NT-3 + NT-4-induced outgrowth. IFNgamma stimulated neurite extension in the presence of NT-3 + NT-4 and NT-3 + NGF. TNFalpha inhibited NT-3-, NT-3 + NGF-, NT-4 + NGF- and NT-3 + NT-4 + NGF-induced outgrowth. These data suggest that inflammation following nerve injury modulates re-innervation via a cytokine/neurotrophin axis.

The culture of hippocampal-entorhinal brain slices is a widely used model for studying neuronal differentiation, axon growth and pathfinding in vitro. The application of tracers (e.g. biocytin) is a well-established method for studying single or multiple neurons and their extensions in this model. For quantifying the growth of high numbers of axons after lesion, however, genetically expressed enhanced green fluorescent protein (EGFP) has proven particularly useful for labeling living axons in vivo and in vitro. Here, we introduce several EGFP-expressing mouse lines which improve the organotypic brain slice model. The questions addressed determine which mouse line to use: beta-actin-EGFP mice for labeling all cells and their extensions; Tau-EGFP mice for labeling the axoplasma; or Thy-1.2-EGFP mice for labeling the axonal membrane. Cocultures of EGFP-positive entorhinal cortex explants with EGFP-negative hippocampal explants allow the monitoring of fluorescent axons growing into the hippocampus in an easily quantifiable manner.

The specification and innervation of cerebral subregions is a complex layer-specific process, primed by region-specific transcription factor expression and axonal guidance cues. In Emx2-/- mice, the hippocampus fails to form a normal dentate gyrus as well as the normal layering of principal neurons in the hippocampus proper. Here, we analyzed the late embryonic and postnatal development of the hippocampal formation and its axonal projections in mice lacking Emx2 expression in vitro. As these mutants die perinatally, we used slice cultures of Emx2 mutant hippocampus to circumvent this problem. In late embryonic Emx2-/- cultivated hippocampi, both the perforant path as well as the distribution of calretinin-positive cells are affected. Traced entorhinal afferents in co-cultures with hippocampus from embryonic Emx2-/- mice terminate diffusely in the prospective dentate gyrus in contrast to the layer-specific termination of co-cultures from wild-type littermates. In addition, in brain slice cultures from null mutants the presumptive dentate gyrus failed to develop its normal cytoarchitecture and mature dentate granule cells, including the lack of their mossy fiber projection. Our data indicate that Emx2 is essential for the terminal differentiation of granular cells and the correct formation of extrinsic and intrinsic hippocampal connections.

Targeting neuronal interleukin-4 signaling is beneficial for clinical progression and axon pathology in different neuroinflammation models. IL-4 empowers axons: Multiple sclerosis (MS) is a neuroinflammatory disorder, and current therapies focus on altering immune activity to reduce symptoms. Vogelaar and colleagues tested the ability of intrathecally applied IL-4, a cytokine typically associated with T helper type 2 responses, to treat established disease in several experimental autoimmune encephalomyelitis (EAE) models. IL-4 treatment led to reduced clinical scores, improved locomotor activity, and diminished axon damage. Somewhat surprisingly, the beneficial effects of IL-4 did not depend on T cell modulation in the chronic disease phase. The receptor for IL-4 was observed in postmortem brain histology of several MS patients, and they demonstrated that IL-4 could act directly on neurons in vitro. They also showed benefits of intranasal IL-4 administration in one of the EAE models, which could be a promising avenue to pursue in the clinic. Ongoing axonal degeneration is thought to underlie disability in chronic neuroinflammation, such as multiple sclerosis (MS), especially during its progressive phase. Upon inflammatory attack, axons undergo pathological swelling, which can be reversible. Because we had evidence for beneficial effects of T helper 2 lymphocytes in experimental neurotrauma and discovered interleukin-4 receptor (IL-4R) expressed on axons in MS lesions, we aimed at unraveling the effects of IL-4 on neuroinflammatory axon injury. We demonstrate that intrathecal IL-4 treatment during the chronic phase of several experimental autoimmune encephalomyelitis models reversed disease progression without affecting inflammation. Amelioration of disability was abrogated upon neuronal deletion of IL-4R. We discovered direct neuronal signaling via the IRS1-PI3K-PKC pathway underlying cytoskeletal remodeling and axonal repair. Nasal IL-4 application, suitable for clinical translation, was equally effective in improving clinical outcome. Targeting neuronal IL-4 signaling may offer new therapeutic strategies to halt disability progression in MS and possibly also neurodegenerative conditions. [ABSTRACT FROM AUTHOR]

During development, axonal projections have a remarkable ability to innervate correct dendritic subcompartments of their target neurons and to form regular neuronal circuits. Altered axonal targeting with formation of synapses on inappropriate neurons may result in neurodevelopmental sequelae, leading to psychiatric disorders. Here we show that altering the expression level of the polysialic acid moiety, which is a developmentally regulated, posttranslational modification of the neural cell adhesion molecule NCAM, critically affects correct circuit formation. Using a chemically modified sialic acid precursor ( N-propyl- d-mannosamine), we inhibited the polysialyltransferase ST8SiaII, the principal enzyme involved in polysialylation during development, at selected developmental time-points. This treatment altered NCAM polysialylation while NCAM expression was not affected. Altered polysialylation resulted in an aberrant mossy fiber projection that formed glutamatergic terminals on pyramidal neurons of the CA1 region in organotypic slice cultures and in vivo. Electrophysiological recordings revealed that the ectopic terminals on CA1 pyramids were functional and displayed characteristics of mossy fiber synapses. Moreover, ultrastructural examination indicated a 'mossy fiber synapse'-like morphology. We thus conclude that homeostatic regulation of the amount of synthesized polysialic acid at specific developmental stages is essential for correct synaptic targeting and circuit formation during hippocampal development. [ABSTRACT FROM AUTHOR]

Phospholipid-mediated signalling on neurons provokes diverse responses such as neurogenesis, pattern formation and neurite remodelling. We have recently uncovered a novel set of molecules in the mammalian brain, named plasticity-related genes (PRGs), which mediate lipid phosphate phosphatase activity and provide evidence for their involvement in mechanisms of neuronal plasticity. Here, we report on a new member of the vertebrate-specific PRG family, which we have named plasticity-related gene-3 (PRG-3). PRG-3 is heavily expressed in the brain and shows a specific expression pattern during brain development where PRG-3 expression is found predominantly in neuronal cell layers and is already expressed at embryonic day 16. In the mature brain, strongest PRG-3 expression occurs in the hippocampus and cerebellum. Overexcitation of neurons induced by kainic acid leads to a transient down-regulation of PRG-3. Furthermore, PRG-3 is expressed on neurite extensions and promotes neurite growth and a spreading-like cell body in neuronal cells and COS-7 cells. In contrast to previously described members of the PRG family, PRG-3 does not perform its function through enzymatic phospholipid degradation. In summary, our findings feature a new member of the PRG family which shows dynamic expression regulation during brain development and neuronal excitation. [ABSTRACT FROM AUTHOR]

Outgrowth of axons in the central nervous system is governed by specific molecular cues. Molecules detected so far act as Iigands that bind to specific receptors. Here, we report a new membrane-associated lipid phosphate phosphatase that we have named plasticity-related gene 1 (PRG-1), which facilitates axonal outgrowth during development and regenerative sprouting. PRG-1 is specifically expressed in neurons and is located in the membranes of outgrowing axons. There, it acts as an ectoenzyme and attenuates phospholipid-induced axon collapse in neurons and facilitates outgrowth in the hippocampus. Thus, we propose a novel mechanism by which axons are able to control phospholipid-mediated signaling and overcome the growthinhibiting, phospholipid-rich environment of the extracellular space. [ABSTRACT FROM AUTHOR]

Focuses on a set of attractive and repellent molecules that show the ability to promote or restrict outgrowth of axons from specific hippocampal input regions. Experimental evidence for molecular guidance cues during hippocampal development; Molecules for the development of hippocampal connections.

Abstract Lesion-induced neuronal plasticity in the adult central nervous system of higher vertebrates appears to be controlled by region- and layer-specific molecules. In this study we demonstrate that membrane-bound hippocampal outgrowth-promoting molecules, as present during the development of the entorhino-hippocampal system and absent or masked in the adult hippocampus, appear 10 days after transection of the perforant pathway. We used an outgrowth preference assay to analyse the outgrowth preference of axons from postnatal entorhinal explants on alternating membrane lanes obtained from hippocampus deafferented from its entorhinal input taken 4, 10, 20, 30 and 80 days post-lesion and from adult control hippocampus. Neurites from the entorhinal cortex preferred to extend axons on hippocampal membranes disconnected from their entorhinal input for 10 days in comparison with membranes obtained from unlesioned adult animals. Membranes obtained from hippocampi disconnected from their entorhinal input for 10 days were equally as attractive for growing entorhinal cortex (EC) axons as membranes from early postnatal hippocampi. Further analysis of membrane properties in an outgrowth length assay showed that entorhinal axons extended significantly longer on stripes of lesioned hippocampal membranes in comparison with unlesioned hippocampal membranes. This effect was most prominent 10 days after lesion, a time point at which axonal sprouting and reactive synaptogenesis are at their peak. Phospholipase treatment of membranes obtained from unlesioned hippocampi of adult animals strongly promoted the outgrowth length of entorhinal axons on these membranes but did not affect their outgrowth preference for deafferented hippocampal membranes. Our results indicate that membrane-bound outgrowth-promoting molecules are reactivated in the adult hippocampus following transection of the perforant pathway, and that neonatal entorhinal axons are able to respond to these molecules.... [ABSTRACT FROM AUTHOR]