Adult Central Nervous System Neurons Research Articles

Moving CNS axon growth and regeneration research into human model systems.

Axon regeneration is limited in the adult mammalian central nervous system (CNS) due to both intrinsic and extrinsic factors. Rodent studies have shown that developmental age can drive differences in intrinsic axon growth ability, such that embryonic rodent CNS neurons extend long axons while postnatal and adult CNS neurons do not. In recent decades, scientists have identified several intrinsic developmental regulators in rodents that modulate growth. However, whether this developmentally programmed decline in CNS axon growth is conserved in humans is not yet known. Until recently, there have been limited human neuronal model systems, and even fewer age-specific human models. Human in vitro models range from pluripotent stem cell-derived neurons to directly reprogrammed (transdifferentiated) neurons derived from human somatic cells. In this review, we discuss the advantages and disadvantages of each system, and how studying axon growth in human neurons can provide species-specific knowledge in the field of CNS axon regeneration with the goal of bridging basic science studies to clinical trials. Additionally, with the increased availability and quality of 'omics datasets of human cortical tissue across development and lifespan, scientists can mine these datasets for developmentally regulated pathways and genes. As there has been little research performed in human neurons to study modulators of axon growth, here we provide a summary of approaches to begin to shift the field of CNS axon growth and regeneration into human model systems to uncover novel drivers of axon growth.

Brain-derived neurotrophic factor released from blood platelets prevents dendritic atrophy of lesioned adult central nervous system neurons.

In humans and other primates, blood platelets contain high concentrations of brain-derived neurotrophic factor due to the expression of the BDNF gene in megakaryocytes. By contrast, mice, typically used to investigate the impact of CNS lesions, have no demonstrable levels of brain-derived neurotrophic factor in platelets, and their megakaryocytes do not transcribe significant levels of the Bdnf gene. Here, we explore potential contributions of platelet brain-derived neurotrophic factor with two well-established CNS lesion models, using 'humanized' mice engineered to express the Bdnf gene under the control of a megakaryocyte-specific promoter. Retinal explants prepared from mice containing brain-derived neurotrophic factor in platelets were labelled using DiOlistics and the dendritic integrity of retinal ganglion cells assessed after 3 days by Sholl analysis. The results were compared with retinas of wild-type animals and with wild-type explants supplemented with saturating concentrations of brain-derived neurotrophic factor or the tropomyosin kinase B antibody agonist, ZEB85. An optic nerve crush was also performed, and the dendrites of retinal ganglion cells similarly assessed 7-day post-injury, comparing the results of mice containing brain-derived neurotrophic factor in platelets with wild-type animals. In mice engineered to contain brain-derived neurotrophic factor in platelets, the mean serum brain-derived neurotrophic factor levels were 25.74 ± 11.36 ng/mL for homozygous and 17.02 ± 6.44 ng/mL for heterozygous mice, close to those determined in primates. Retinal explants from these animals showed robust preservation of dendrite complexity, similar to that seen with wild-type explants incubated with medium supplemented with brain-derived neurotrophic factor or the tropomyosin receptor kinase B antibody agonist, ZEB85. The Sholl areas under curve were 1811 ± 258, 1776 ± 435 and 1763 ± 256 versus 1406 ± 315 in the wild-type control group (P ≤ 0.001). Retinal ganglion cell survival based on cell counts was similar in all four groups, showing ∼15% loss. A robust neuroprotective effect was also observed following optic nerve crush when assessing the dendrites of the retinal ganglion cells in the transgenic mouse, with Sholl area under the curve significantly higher compared to wild-type (2667 ± 690 and 1921 ± 392, P = 0.026), with no significant difference in the contralateral eye controls. Repeat experiments found no difference in cell survival, with both showing ∼50% loss. These results indicate that platelet brain-derived neurotrophic factor has a strong neuroprotective effect on the dendrite complexity of retinal ganglion cells in both an ex vivo and in vivo model, suggesting that platelet brain-derived neurotrophic factor is likely to be a significant neuroprotective factor in primates.

Methods for culturing adult CNS neurons reveal a CNS conditioning effect

SummaryNeuronal cultures provide a basis for reductionist insights that rely on molecular and pharmacological manipulation. However, the inability to culture mature adult CNS neurons limits our understanding of adult neuronal physiology. Here, we report methods for culturing adult central nervous system neurons in large numbers and across multiple brain regions for extended time periods. Primary adult neuronal cultures develop polarity; they establish segregated dendritic and axonal compartments, maintain resting membrane potentials, exhibit spontaneous and evoked electrical activity, and form neural networks. Cultured adult neurons isolated from different brain regions such as the hippocampus, cortex, brainstem, and cerebellum exhibit distinct cell morphologies, growth patterns, and spontaneous firing characteristics reflective of their regions of origin. Using adult motor cortex cultures, we identify a CNS “conditioning” effect after spinal cord injury. The ability to culture adult neurons offers a valuable tool for studying basic and therapeutic science of the brain.

Bio-C (Modified Hyaluronic Acid-Coated-Collagen Tube) Implants Enable Functional Recovery after Complete Spinal Cord Injury.

Neural repair within the central nervous system (CNS) has been extremely challenging due to limited abilities of adult CNS neurons to regenerate, particularly in a highly inflammatory injury environment that is also filled with myelin debris. Spinal cord injury (SCI) is a serious medical condition that often leads to paralysis and currently has no effective treatment. Here we report the construction of a novel biocompatible and biodegradable material, Bio-C, through coating of acid-desalted-collagen (ADC) tube with pre-modified hyaluronic acid, which, after implantation, can elicit quite robust neural regeneration and functional recovery after complete spinal-cord transection with a 2 mm–spinal-cord-segment removal in mice. We combined morphological, electrophysiological, and objective transcriptomic analyses, in addition to behavioral analyses, to demonstrate neural tissue regeneration and functional recovery through the establishment of Bio-C-induced anti-inflammatory, neurogenic, and neurotrophic microenvironment. Through this study, we unveiled the underlying logic for CNS neural repair.

K+/Cl- co-transporter-2 upmodulation: a multi-modal therapy to treat spinal cord injury.

K+/Cl- co-transporter-2 upmodulation: a multi-modal therapy to treat spinal cord injury.

Brain delivering RNA-based therapeutic strategies by targeting mTOR pathway for axon regeneration after central nervous system injury.

Injuries to the central nervous system (CNS) such as stroke, brain, and spinal cord trauma often result in permanent disabilities because adult CNS neurons only exhibit limited axon regeneration. The brain has a surprising intrinsic capability of recovering itself after injury. However, the hostile extrinsic microenvironment significantly hinders axon regeneration. Recent advances have indicated that the inactivation of intrinsic regenerative pathways plays a pivotal role in the failure of most adult CNS neuronal regeneration. Particularly, substantial evidence has convincingly demonstrated that the mechanistic target of rapamycin (mTOR) signaling is one of the most crucial intrinsic regenerative pathways that drive axonal regeneration and sprouting in various CNS injuries. In this review, we will discuss the recent findings and highlight the critical roles of mTOR pathway in axon regeneration in different types of CNS injury. Importantly, we will demonstrate that the reactivation of this regenerative pathway can be achieved by blocking the key mTOR signaling components such as phosphatase and tensin homolog (PTEN). Given that multiple mTOR signaling components are endogenous inhibitory factors of this pathway, we will discuss the promising potential of RNA-based therapeutics which are particularly suitable for this purpose, and the fact that they have attracted substantial attention recently after the success of coronavirus disease 2019 vaccination. To specifically tackle the blood-brain barrier issue, we will review the current technology to deliver these RNA therapeutics into the brain with a focus on nanoparticle technology. We will propose the clinical application of these RNA-mediated therapies in combination with the brain-targeted drug delivery approach against mTOR signaling components as an effective and feasible therapeutic strategy aiming to enhance axonal regeneration for functional recovery after CNS injury.

Actin dynamics in the growth cone: a key player in axon regeneration.

Neuronal development, maintenance and function depends on the tight regulation of cytoskeleton organization and dynamics. Following injury, adult central nervous system neurons have a limited ability to regenerate and to recapitulate their robust developmental axon growth. This decreased regenerative capacity is set by their inability to establish regeneration-competent growth cones. Growth cones are actin-enriched structures that regulate axon extension rate and direction. During neuronal development, increasing actin dynamics in the growth cone through the regulation of the activity of specific actin-binding proteins leads to increased axon elongation. Here, we will focus on recent findings showing that enhanced axon regeneration in the adult nervous system can be achieved by promoting actin dynamics, or by decreasing actomyosin contraction in the growth cone. These discoveries underscore the importance of actin organization in the growth cone as a key factor to promote axon (re)growth that should be further explored in the future.

Stabilization of primary cilia reduces abortive cell cycle re-entry to protect injured adult CNS neurons from apoptosis.

Abortive cell cycle (ACC) re-entry of apoptotic neurons is a recently characterized phenomenon that occurs after central nervous system (CNS) injury or over the course of CNS disease. Consequently, inhibiting cell cycle progression is neuroprotective in numerous CNS pathology models. Primary cilia are ubiquitous, centriole-based cellular organelles that prevent cell cycling, but their ability to modulate abortive cell cycle has not been described. Here, we show that neuronal cilia are ablated in-vitro and in-vivo following injury by hypoxia or optic nerve transection (ONT), respectively. Furthermore, forced cilia resorption sensitized neurons to these injuries and enhanced cell death. In contrast, pharmacological inhibition or shRNA knockdown of the proteins that disassemble the cilia increased neuron survival and decreased the phosphorylation of retinoblastoma (Rb), a master switch for cell cycle re-entry. Our findings show that the stabilization of neuronal primary cilia inhibits, at least transiently, apoptotic cell cycling, which has implications for future therapeutic strategies that halt or slow the progression of neurodegenerative diseases and acute CNS injuries.

Recent advances in the use of magnetic nanoparticles to promote neuroregeneration.

Recent advances in the use of magnetic nanoparticles to promote neuroregeneration.

Systemic administration of epothilone D improves functional recovery of walking after rat spinal cord contusion injury

Central nervous system (CNS) injuries cause permanent impairments of sensorimotor functions as mature neurons fail to regenerate their severed axons. The poor intrinsic growth capacity of adult CNS neurons and the formation of an inhibitory lesion scar are key impediments to axon regeneration. Systemic administration of the microtubule stabilizing agent epothilone B promotes axon regeneration and recovery of motor function by activating the intrinsic axonal growth machinery and by reducing the inhibitory fibrotic lesion scar. Thus, epothilones hold clinical promise as potential therapeutics for spinal cord injury. Here we tested the efficacy of epothilone D, an epothilone B analog with a superior safety profile. By using liquid chromatography and mass spectrometry (LC/MS), we found adequate CNS penetration and distribution of epothilone D after systemic administration, confirming the suitability of the drug for non-invasive CNS treatment. Systemic administration of epothilone D reduced inhibitory fibrotic scarring, promoted regrowth of injured raphespinal fibers and improved walking function after mid-thoracic spinal cord contusion injury in adult rats. These results confirm that systemic administration of epothilones is a valuable therapeutic strategy for CNS regeneration and repair after injury and provides a further advance for potential clinical translation.

The expression of chemorepulsive guidance receptors and the regenerative abilities of spinal-projecting neurons after spinal cord injury

Spinal cord injury (SCI) in mammals leads to permanent loss of function because axons do not regenerate in the central nervous system (CNS). To date, treatments based on neutralizing inhibitory environmental cues, such as the myelin-associated growth inhibitors and chondroitin sulfate proteoglycans, or on adding neurotrophic factors, have had limited success in enhancing regeneration. Published studies suggested that multiple axon guidance cues (repulsive guidance molecule (RGM) family, semaphorins, ephrins, and netrins) persist in adult animals, and that their expression is upregulated after CNS injury. Moreover, many adult CNS neurons continue to express axon guidance receptors. We used the advantages of the lamprey CNS to test the hypotheses that the regenerative abilities of spinal-projecting neurons depend upon their expression of chemorepulsive guidance receptors. After complete spinal transection, lampreys recover behaviorally, and injured axons grow selectively in their correct paths. However, the large identified reticulospinal (RS) neurons in the lamprey brain are heterogeneous in their regenerative abilities – some are high regeneration capacity neurons (probability of axon regeneration >50%), others are low regeneration capacity neurons (<30%). Here we report that the RGM receptor Neogenin is expressed preferentially in the low regeneration capacity RS neurons that regenerate poorly, and that downregulation of Neogenin by morpholino antisense oligonucleotides enhances regeneration of RS axons after SCI. Moreover, lamprey CNS neurons co-express multiple guidance receptors (Neogenin, UNC5 and PlexinA), suggesting that the regenerative abilities of spinal-projecting neurons might reflect the summed influences of the chemorepulsive guidance receptors that they express.

Epigenetic profiling reveals a developmental decrease in promoter accessibility during cortical maturation in vivo

Axon regeneration in adult central nervous system (CNS) is limited in part by a developmental decline in the ability of injured neurons to re-express needed regeneration associated genes (RAGs). Adult CNS neurons may lack appropriate pro-regenerative transcription factors, or may display chromatin structure that restricts transcriptional access to RAGs. Here we performed epigenetic profiling around the promoter regions of key RAGs, and found progressive restriction across a time course of cortical maturation. These data identify a potential intrinsic constraint to axon growth in adult CNS neurons. Neurite outgrowth from cultured postnatal cortical neurons, however, proved insensitive to treatments that improve axon growth in other cell types, including combinatorial overexpression of AP1 factors, overexpression of histone acetyltransferases, and pharmacological inhibitors of histone deacetylases. This insensitivity could be due to intermediate chromatin closure at the time of culture, and highlights important differences in cell culture models used to test potential pro-regenerative interventions.

The tumor suppressor HHEX inhibits axon growth when prematurely expressed in developing central nervous system neurons.

Neurons in the embryonic and peripheral nervous system respond to injury by activating transcriptional programs supportive of axon growth, ultimately resulting in functional recovery. In contrast, neurons in the adult central nervous system (CNS) possess a limited capacity to regenerate axons after injury, fundamentally constraining repair. Activating pro-regenerative gene expression in CNS neurons is a promising therapeutic approach, but progress is hampered by incomplete knowledge of the relevant transcription factors. An emerging hypothesis is that factors implicated in cellular growth and motility outside the nervous system may also control axon growth in neurons. We therefore tested sixty-nine transcription factors, previously identified as possessing tumor suppressive or oncogenic properties in non-neuronal cells, in assays of neurite outgrowth. This screen identified YAP1 and E2F1 as enhancers of neurite outgrowth, and PITX1, RBM14, ZBTB16, and HHEX as inhibitors. Follow-up experiments are focused on the tumor suppressor HHEX, one of the strongest growth inhibitors. HHEX is widely expressed in adult CNS neurons, including corticospinal tract neurons after spinal injury, but is present only in trace amounts in immature cortical neurons and adult peripheral neurons. HHEX overexpression in early postnatal cortical neurons reduced both initial axonogenesis and the rate of axon elongation, and domain deletion analysis strongly implicated transcriptional repression as the underlying mechanism. These findings suggest a role for HHEX in restricting axon growth in the developing CNS, and substantiate the hypothesis that previously identified oncogenes and tumor suppressors can play conserved roles in axon extension.

The necessary role of mTORC1 in central nervous system axon regeneration.

Permanent loss of vital functions after central nervous system (CNS) injury, e.g., blindness in traumatic optic nerve (ON) injury or paralysis in spinal cord injury, occurs in part because axons in the adult mammalian CNS do not regenerate after injury. Growth failure is due to the diminished intrinsic regenerative capacity of mature neurons and the inhibitory environment of the adult CNS. Neutralizing extracellular inhibitory molecules genetically or pharmacologically yields only limited regeneration and functional recovery, highlighting the critical importance of neuron-intrinsic factors. To explore the intrinsic regenerative signaling molecules, a relatively simple but robust in vivo model that replicates CNS traumatic injury and permits straightforward interpretation is required. Mouse retinal ganglion cell (RGC) and ON provide a valuable in vivo neural injury system to study intrinsic growth mechanisms in adult CNS neurons (Figure 1). Retina and ON are CNS structures that are essential for visual functions. RGC is the only neuronal type to relay visual information from retina to brain. The ON is formed by the projection axons sent exclusively from RGCs; it has the simplicity of a unidirectional axon pathway, which insures that any nerve fibers observed distal to a complete crush injury have regenerated and do not represent spared axons that underwent collateral sprouting or efferent axons from the brain to the retina. Its easy access allows adeno-associated viruses to be injected directly into the vitreous chamber of the eye and express transgenes specifically and efficiently in adult RGCs. This spatially and temporally controlled genetic manipulation allows us to overcome developmental issues associated with germ line manipulation and to test interventions that can potentially be translated to therapies. Exploiting the anatomical and technical advantages of the RGC/ON crush model to understand the intrinsic mechanisms of regenerative failure led us to the finding that deletion of phosphatase and tensin homolog (PTEN) promoted significant ON regeneration (Park et al., 2008). PTEN, a lipid phosphatase, is a major negative regulator of the phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin complex 1 (mTORC1) pathway. Similar axon regeneration phenotypes after PTEN deletion have been reported for mouse cortical motor neurons (Liu et al., 2010), drosophila sensory neurons (Song et al., 2012) and C. elegans motor neurons (Byrne et al., 2014), presumably through activating PI3K-mTORC1-controlled cell growth (Figure 2). Direct activation of mTORC1 also promotes axon regeneration in dopaminergic neurons (Kim et al., 2011) and peripheral nerves (Abe et al., 2010), further support for the critical role of mTORC1 in axon regeneration.

Chondroitin sulfate glycosaminoglycans for CNS homeostasis-implications for material design.

Chondroitin sulfate proteoglycans (CSPGs) are complex biomolecules that are known to facilitate patterning of axonal direction and cell migration during the early growth and development phase of the mammalian central nervous system (CNS). In adults, they continue to control neuronal plasticity as major constituents of the "peri-neuronal nets" (PNNs) that surround adult CNS neurons. CSPGs are also barrier-forming molecules that are selectively upregulated by invading reactive astroglia after injury to the CNS, and are responsible for the active repulsion of regenerating neurons post-injury. Recent evidence however suggests that the diverse sulfated glycosaminoglycan (GAG) side chains attached to CSPGs are key components that play paradoxical roles in influencing nerve regeneration post-injury to the CNS. Sulfated GAG repeats attached to the CSPG core protein help mediate cell migration, neuritogenesis, axonal pathfinding, and axonal repulsion by directly trapping and presenting a whole host of growth factors to cells locally, or by binding to specific membrane bound proteins on the cell surface to influence cellular function. In this review, we will present the current gamut of interventional strategies used to bridge CNS deficits, and discuss the potential advantages of using sulfated GAG based biomaterials to facilitate the repair and regeneration of the injured CNS.

Microfluidic systems for axonal growth and regeneration research.

Damage to the adult mammalian central nervous system (CNS) often results in persistent neurological deficits with limited recovery of functions. The past decade has seen increasing research efforts in neural regeneration research with the ultimate goal of achieving functional recovery. Many studies have focused on prevention of further neural damage and restoration of functional connections that are compromised after injury or pathological damage. Compared to the peripheral nervous system, the failure of the adult CNS to regenerate is largely attributed to two basic aspects: inhibitory environmental influences and decreased growth capabilities of adult CNS neurons. Since early demonstration of successful growth of injured CNS axons into grafted peripheral nerve (David and Aguayo, 1981), multiple CNS axonal growth inhibitory factors have been identified and are mainly associated with degenerating CNS myelin (such as Nogo, MAG, OMgp) and with glial scar (such as chondroitin sulfate proteoglycans, CSPGs) (Yiu and He, 2006). However, blockade of these extracellular inhibitory signals alone is often insufficient for the majority of injured axons to achieve long-distance regeneration, as intrinsic regenerative capacity of mature CNS neurons is also a critical determinant for axon re-growth(Sun et al., 2011). Combinatory strategies that enhance neuronal growth and in the meantime overcome environmental inhibitory cues appear to confer better axonal regeneration and neural repair (Wang et al., 2012). While animal models are instrumental and indispensable to our understanding of CNS responses to injury and investigation of intervention strategies and functional regeneration, in vitro models have been designed to address specific and unique questions due to their accessibilities to experimental manipulations and relatively low cost. For instance, early findings from in vitro culture experiments formed the basis for the concept of CNS myelin-associated inhibitory molecules such as Nogo (Schwab and Thoenen, 1985; Chen et al., 2000; GrandPre et al., 2000). Attempts to identify compounds that overcome the inhibition of CNS myelin and CSPGs on neurite outgrowth in culture have revealed key neuronal signaling components that mediate the inhibitory effects of myelin and CSPGs (Sivasankaran et al., 2004). However, conventional culture system often has to use neuronal cultures at low cell density and for only a short period of time due to technical difficulties in monitoring and quantifying axonal growth. To develop effective CNS regenerative strategies, fast and reliable assessment of axonal growth would be critical not only to identify and select potentially interesting candidate molecules that promote axon extension over inhibitory molecules, but also to rule out poor ones. Equally important are considerations that neurons are highly polarized cells and that damaged axon could be extending far away from the cell body and encountering drastically different microenvironment than that of the soma. As such, signaling events elicited by extrinsic factors are most likely spatially regulated and may have different functional outputs. Over the years, compartmentalized neuronal culture systems have been developed. Now the emergence of microfluidics technology offers many advantages and versatilities for axonal growth and regeneration studies (Figure 1). Figure 1 Schematic illustrations of compartmentalized neuron culture platforms for axon isolation.

The insulin-like growth factor 1 receptor is essential for axonal regeneration in adult central nervous system neurons.

Axonal regeneration is an essential condition to re-establish functional neuronal connections in the injured adult central nervous system (CNS), but efficient regrowth of severed axons has proven to be very difficult to achieve. Although significant progress has been made in identifying the intrinsic and extrinsic mechanisms involved, many aspects remain unresolved. Axonal development in embryonic CNS (hippocampus) requires the obligate activation of the insulin-like growth factor 1 receptor (IGF-1R). Based on known similarities between axonal growth in fetal compared to mature CNS, we decided to examine the expression of the IGF-1R, using an antibody to the βgc subunit or a polyclonal anti-peptide antibody directed to the IGF-R (C20), in an in vitro model of adult CNS axonal regeneration, namely retinal ganglion cells (RGC) derived from adult rat retinas. Expression of both βgc and the β subunit recognized by C20 antibody were low in freshly isolated adult RGC, but increased significantly after 4 days in vitro. As in embryonic axons, βgc was localised to distal regions and leading growth cones in RGC. IGF-1R-βgc co-localised with activated p85 involved in the phosphatidylinositol-3 kinase (PI3K) signaling pathway, upon stimulation with IGF-1. Blocking experiments using either an antibody which neutralises IGF-1R activation, shRNA designed against the IGF-1R sequence, or the PI3K pathway inhibitor LY294002, all significantly reduced axon regeneration from adult RGC in vitro (∼40% RGC possessed axons in controls vs 2–8% in the different blocking studies). Finally, co-transfection of RGC with shRNA to silence IGF-1R together with a vector containing a constitutively active form of downstream PI3K (p110), fully restored axonal outgrowth in vitro. Hence these data demonstrate that axonal regeneration in adult CNS neurons requires re-expression and activation of IGF-1R, and targeting this system may offer new therapeutic approaches to enhancing axonal regeneration following trauma.

Identification of the role of C/EBP in neurite regeneration following microarray analysis of a L. stagnalisCNS injury model

BackgroundNeuronal regeneration in the adult mammalian central nervous system (CNS) is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. In contrast, the CNS of adult Lymnaea stagnalis (L. stagnalis), a freshwater pond snail, is capable of spontaneous regeneration following neuronal injury. Thus, L. stagnalis has served as an animal model to study the cellular mechanisms underlying neuronal regeneration. However, the usage of this model has been limited due to insufficient molecular tools. We have recently conducted a partial neuronal transcriptome sequencing project and reported over 10,000 EST sequences which allowed us to develop and perform a large-scale high throughput microarray analysis.ResultsTo identify genes that are involved in the robust regenerative capacity observed in L. stagnalis, we designed the first gene chip covering ~15, 000 L. stagnalis CNS EST sequences. We conducted microarray analysis to compare the gene expression profiles of sham-operated (control) and crush-operated (regenerative model) central ganglia of adult L. stagnalis. The expression levels of 348 genes were found to be significantly altered (p < 0.05) following nerve injury. From this pool, 67 sequences showed a greater than 2-fold change: 42 of which were up-regulated and 25 down-regulated. Our qPCR analysis confirmed that CCAAT enhancer binding protein (C/EBP) was up-regulated following nerve injury in a time-dependent manner. In order to test the role of C/EBP in regeneration, C/EBP siRNA was applied following axotomy of cultured Lymnaea PeA neurons. Knockdown of C/EBP following axotomy prevented extension of the distal, proximal and intact neurites. In vivo knockdown of C/EBP postponed recovery of locomotory activity following nerve crush. Taken together, our data suggest both somatic and local effects of C/EBP are involved in neuronal regeneration.ConclusionsThis is the first high-throughput microarray study in L. stagnalis, a model of axonal regeneration following CNS injury. We reported that 348 genes were regulated following central nerve injury in adult L. stagnalis and provided the first evidence for the involvement of local C/EBP in neuronal regeneration. Our study demonstrates the usefulness of the large-scale gene profiling approach in this invertebrate model to study the molecular mechanisms underlying the intrinsic regenerative capacity of adult CNS neurons.

Nogo-66 receptor activation inhibits neurite outgrowth and increases β-amyloid protein secretion of cortical neurons

A Nogo-A to Nogo-66 receptor (NgR) pathway is well known to contribute to the inhibition of the neurite regeneration of adult central nervous system neurons after traumatic injuries. Recent evidence suggests that Nogo-A and NgR are involved in the pathology of Alzheimer's disease (AD), as evidenced by the fact that Nogo-A is overexpressed by hippocampal neurons in patients with AD and is associated with β-amyloid protein (Aβ) deposits in senile plaques. In the present experiments, we investigated the potential role of Nogo-A in both neurite outgrowth and Aβ generation in cortical neurons. Our results showed that activation of NgR not only inhibited neurite outgrowth in cortical neurons by activating the rho-associated coiled coil-containing protein kinase (ROCK) and protein kinase C, but also promoted their Aβ secretion, which was at least in part activated by ROCK. These findings suggest that the overexpression of Nogo-A and the activation of NgR inhibit neurite outgrowth and alter neuronal metabolism, resulting in overproduction and/or release of Aβ, which in turn may trigger the onset and development of AD. Inhibition of ROCK can promote neurite outgrowth and reduce Aβ production of cortical neuron, which suggests that ROCK appears to be a good target for AD therapy.

Distinct Expression of c-Jun and HSP27 in Axotomized and Spared Bulbospinal Neurons After Cervical Spinal Cord Injury

In several populations of adult central nervous system neurons, axon damage can lead to an up-regulation of some transcription factors among which is c-Jun, known to be a key regulator of neuron cell body response to injury and of its intrinsic potential for axon regeneration. Thus, cervical spinal hemisection leads to c-Jun up-regulation in bulbospinal and rubrospinal axotomized neurons. The aims of the present study were to specify, after a unilateral cervical spinal cord injury, the expression of another marker of the neuronal stress response, heat shock protein 27 (HSP27) in axotomized neurons of the medulla (labeled by fluorogold retrograde tracer), and to compare it to that of c-Jun. In the medulla of injured rats, HSP27 and phospho-HSP27 were expressed in sub-populations of axotomized neurons, principally in the rostral ventral respiratory group (rVRG) (20%), the dorsal part of the gigantocellularis (Gi) (50%), and vestibular nucleus, but seldom in the ventral Gi and raphe nucleus, indicating a heterogeneous post-lesion cell body response between these different neuron populations. By contrast, phospho-c-Jun was up-regulated in axotomized neurons in all nuclei containing bulbospinal neurons, including the rVRG and Gi. In these areas, phospho-c-Jun was also up-regulated in uninjured bulbospinal neurons which project caudal to the spinal cord injury (labeled by fluorogold retrograde tracer). In contrast to phospho-c-Jun, HSP27 immunoreactivity was generally not present in neurons with spared axons. Our results show that various bulbospinal neuron populations react differentially to the injury and that spinal cord injury affects also bulbospinal neurons that are spared by the injury. However, the molecular cell body response of spared neurons is distinct from that of axotomized neurons since they can up-regulate c-Jun but not HSP27.