GAP-43 mRNA Research Articles

Voluntary exercise increases axonal regeneration from sensory neurons.

Recent advances in understanding the role of neurotrophins on activity-dependent plasticity have provided insight into how behavior can affect specific aspects of neuronal biology. We present evidence that voluntary exercise can prime adult dorsal root ganglion neurons for increased axonal regeneration through a neurotrophin-dependent mechanism. Dorsal root ganglion neurons showed an increase in neurite outgrowth when cultured from animals that had undergone 3 or 7 days of exercise compared with sedentary animals. Neurite length over 18-22 h in culture correlated directly with the distance that animals ran. The exercise-conditioned animals also showed enhanced regrowth of axons after an in vivo nerve crush injury. Sensory ganglia from the 3- and 7-day-exercised animals contained higher brain-derived neurotrophic factor, neurotrophin 3, synapsin I, and GAP43 mRNA levels than those from sedentary animals. Consistent with the rise in brain-derived neurotrophic factor and neurotrophin 3 during exercise, the increased growth potential of the exercise-conditioned animals required activation of the neurotrophin signaling in vivo during the exercise period but did not require new mRNA synthesis in culture.

Axonal reinjury reveals the survival and re-expression of regeneration-associated genes in chronically axotomized adult mouse motoneurons

Recently, we reported that chronically axotomized rubrospinal neurons survive for up to 1 year in an atrophied state. This finding contrasted previous work suggesting the death of up to 50% of the neurons over time. In the adult mouse, the majority of facial motoneurons appear to be lost as a result of chronic nerve resection. Here, we sought to determine if chronically resected adult mouse facial motoneurons, like rubrospinal neurons, survive in an atrophied state. To test this hypothesis, we asked whether a second nerve injury, 10 weeks after an initial nerve resection, could stimulate a regenerative cell body response. After chronic resection (10 weeks), mouse facial motoneurons underwent atrophy resulting in a loss of countable neuronal cell bodies. In addition, the motoneurons failed to maintain their initial increase in expression of GAP-43 and α-tubulin mRNA. Reinjury of 10-week chronically resected facial motoneurons by the removal of the neuroma reversed the atrophy of the cell bodies and increased the percentage of identifiable cell bodies from 36% of contralateral to 79% in C57BL/6-C3H mice and from 28% of contralateral to 40% in Balb/c mice. Moreover, the reinjured motoneurons displayed an increase in GAP-43 and α-tubulin mRNA expression. The results of this study indicate that a second axon injury stimulates regenerative cell body responses in chronically resected mouse facial motoneurons and suggest previous studies using this model may have overestimated the number of dying motoneurons.

The use of GAP-43 mRNA quantification in high throughput screening of putative neuroprotective agents in dorsal root ganglion cultures

Large scale screening for neuroprotective drugs for peripheral neuropathies requires development of a high throughput system that is reliable and reproducible. Currently most accurate outcome measures of axonal degeneration are based on time-consuming, laborious measurement of morphological changes in neurites. In order to improve on the scalability of the screening procedure we developed a real-time RT–PCR based method of gene expression that correlates very well with morphological measures of neuritic degeneration. We examined the changes in GAP-43 expression in primary dorsal root ganglion (DRG) neurons in vitro with exposure to a zalcitabine (ddC), an antiretroviral drug that causes neuropathy in human immunodeficiency virus (HIV)-infected individuals, with and without FK506, an immunophilin ligand with neuroprotective and neuroregenerative properties. Similar to morphological measures of neuritic degeneration, in ddC-treated cultures there was a reduction in the expression of GAP-43 mRNA. This was prevented, in a dose-dependent manner, by co-administration of FK506. This assay, performed in a 96-well format, can easily be scaled for high throughput screening (HTS) using robotic systems.

Increase of the RNA-binding protein HuD and posttranscriptional up-regulation of the GAP-43 gene during spatial memory.

Neuronal ELAV-like proteins (HuB, HuC, and HuD) are highly conserved RNA-binding proteins able to selectively associate with the 3' UTR of a subset of target mRNAs and increase their cytoplasmic stability and rate of translation. We previously demonstrated the involvement of these proteins in learning, reporting that they undergo a sustained up-regulation in the hippocampus of mice trained in a spatial discrimination task. Here, we extend this finding, showing that a similar up-regulation occurs in the hippocampus of rats trained in another spatial learning paradigm, the Morris water maze. HuD, a strictly neuron-specific ELAV-like protein, is shown to increase after learning, with a preferential binding to the cytoskeletal fraction. HuD up-regulation is associated with an enhancement of GAP-43 mRNA and protein levels, with an apparently increased HuD colocalization with the GAP-43 mRNA and an increased association of neuronal ELAV-like proteins with the GAP-43 mRNA. These learning-dependent biochemical events appear to be spatiotemporally controlled, because they do not occur in another brain region involved in learning, the retrosplenial cortex, and at the level of protein expression they show extinction 1 month after training despite memory retention. By contrast, HuD mRNA levels still remain increased after 1 month in the CA1 region. This persistence may have implications for long-term memory recall.

Corticothalamic cells in layers 5 and 6 of primary and secondary sensory cortex express GAP-43 mRNA in the adult rat.

The expression of a presynaptic phosphoprotein, growth-associated protein (GAP)-43, is associated with synaptogenesis during development and synaptic remodeling in the adult. This study examined GAP-43 mRNA expression and distribution in primary and secondary areas of visual, auditory, and somatosensory cortex of the adult rat, by in situ hybridization with a digoxigenin-coupled mRNA probe, focusing particularly on the corticothalamic cells in layers 5 and 6. In the six cortical areas studied, GAP-43 mRNA was expressed predominantly in layers 5 and 6 and was greater in secondary than primary areas. There were densely labeled cells in layers 5 and 6 of all areas, which showed a restricted sublaminar distribution in primary areas and more even distribution in secondary areas. Combining retrograde transport of rhodamine beads with in situ hybridization in visual and auditory cortex showed that corticothalamic cells in layers 5 and 6 express GAP-43 mRNA. There are more of these GAP-43 mRNA positive corticothalamic cells in layer 5 of secondary areas than in primary areas. The evidence suggests that in the adult rat, plasticity related to GAP-43 is present in primary and secondary sensory cortex and more so in secondary areas.

βIi-tubulin and GAP 43 mRNA expression in chronically injured neurons of the red nucleus after a second spinal cord injury

Regeneration by chronically injured supraspinal neurons is enhanced by treatment of a spinal cord lesion site with a variety of neurotrophic and growth factors. The removal of scar tissue, with subsequent reinjury of the spinal cord, is necessary for injured axons to access tissue transplants placed into the lesion to support axon regrowth. The present study examined chronically injured and reinjured rubrospinal tract (RST) neurons to determine if changes in gene expression could explain the failure of these neurons to regenerate without exogenous trophic factor support. Adult female rats were subjected to a right full hemisection lesion via aspiration of the cervical level 3 spinal cord. Using radioactive cDNA probes and in situ hybridization, RST neurons in the contralateral red nucleus were examined for changes in mRNA levels of βII-tubulin and GAP 43 in an acute injury period (6 h–3 days), a chronic injury period (28 days after spinal cord injury (SCI)) and following a second lesion of the chronic injury site (6 h–7 days). Based upon the analysis of gene expression in single cells, GAP-43 mRNA levels were increased as early as 1 day following the initial SCI, but were no different than uninjured control levels at 28 days postoperative (dpo). The response to relesion was more rapid and higher than that detected after the initial injury with a significant increase in GAP 43 mRNA at 6 h that was maintained for at least 7 days. βII-tubulin mRNA levels remained unchanged until 3 days after an acute injury followed by a decrease in expression to 30% below uninjured control values at 28 dpo. The expression of βII-tubulin mRNA was significantly higher within 6 h after a second injury, where it remained stable for 5 days before a second increase occurred at 7 days after reinjury of the spinal cord. Thus, neurons in a chronic injury state retain the ability to respond to a traumatic injury and, in fact, neurons subjected to a second injury exhibit a significantly heightened expression of regeneration-associated genes.

Increased expression and localization of the RNA-binding protein HuD and GAP-43 mRNA to cytoplasmic granules in DRG neurons during nerve regeneration☆

The neuronal-specific RNA-binding protein, HuD, binds to a U-rich regulatory element of the 3′ untranslated region (3′ UTR) of the GAP-43 mRNA and delays the onset of its degradation. We have recently shown that overexpression of HuD in embryonic rat cortical cells accelerated the time course of normal neurite outgrowth and resulted in a twofold increase in GAP-43 mRNA levels. Given this evidence, we sought to investigate the involvement of HuD during nerve regeneration. It is known that HuD protein and GAP-43 mRNA are expressed in the dorsal root ganglia (DRG) of adult rat and that GAP-43 is upregulated in DRG neurons during regeneration. In this study, we examined the expression patterns and levels of HuD and GAP-43 mRNA in DRG neurons following sciatic nerve injury using a combination of in situ hybridization, immunocytochemistry, and quantitative RT-PCR. GAP-43 and HuD expression increased in the ipsilateral DRG during the first 3 weeks of regeneration, with peak values seen at 7 days postcrush. At this time point, the levels of HuD and GAP-43 mRNAs in the ipsilateral DRG increased by twofold and sixfold, respectively, relative to the contralateral DRG. Not only were the temporal patterns of expression of HuD protein and GAP-43 mRNA similar, but also they were found to colocalize in the cytoplasm of DRG neurons. Moreover, both molecules were distributed in cytoplasmic granules containing ribosomal RNA. In conclusion, our results suggest that HuD is involved in the upregulation of GAP-43 expression observed at early stages of peripheral nerve regeneration.

GAP-43 mRNA detection by in situ hybridization, direct and indirect in situ RT-PCR in hippocampal and cerebellar tissue sections of adult rat brain

The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that is expressed at high levels during development and axonal growth. To evaluate the cellular distribution of GAP-43 mRNA in the hippocampus and cerebellum of adult rats we applied in situ hybridization (ISH) as well as direct and indirect in situ RT-PCR using biotin as a reporter molecule. ISH resulted in a positive signal in most cerebellar granular cells and in 30% of hippocampal CA3 neurons. Direct in situ RT-PCR yielded cells with strong signals in every region investigated, with elevated background levels most likely related to incorporation of labeled nucleotides into non-specific amplicons through internal priming and DNA repair activity. Indirect in situ RT-PCR turned out to be the best approach for detecting GAP-43 mRNA positive cells. Cerebellar cells exhibiting a positive signal for GAP-43 mRNA were of the granular cell type (98%). Hippocampal neurons with a positive reaction for GAP-43 mRNA included all the neuron groups analyzed, namely CA1 (99%) and CA3 pyramidal cells (94%) and dentate gyrus granule cells (92%). Dentate gyrus granule cells have not tested positive for GAP-43 mRNA detection by molecular morphology analysis. These data show that in normal rats GAP-43 mRNA is present in different cell populations of hippocampal formation, supporting the role of this protein in the ongoing processes of synaptic plasticity.

Transient downregulation of sema3a mrna in a rat model for temporal lobe epilepsy: A novel molecular event potentially contributing to mossy fiber sprouting

Mossy fiber sprouting (MFS) in the hippocampal dentate gyrus is thought to play a critical role in the hyperexcitability of the hippocampus in temporal lobe epilepsy patients. The composition of molecular signals that is needed to direct this sprouting response has not yet been elucidated to a great extent. In the present study we investigated the expression profile of Sema3A mRNA and the axonal growth-associated protein GAP-43 mRNA during the process of electrically induced epileptogenesis in rats. Sema3A is an axon guidance molecule with repellent activity on dentate granule cell axons. It is produced by neurons in the entorhinal cortex, which synapse on the dendrites of dentate granule cells. Upregulation of GAP-43 expression in granule cells has often been reported in conjunction with MFS. After induction of status epilepticus, the expression of Sema3A mRNA was temporarily downregulated in the entorhinal cortex concomitantly with an upregulation of GAP-43 mRNA in dentate granule cells. In the following days, robust MFS into the dentate molecular layer was observed. When the induction of status epilepticus was incomplete the two responses appeared to dissociate, i.e., the downregulation of Sema3A mRNA did not occur, while upregulation of GAP-43 mRNA in dentate granule cells was still displayed. However, in these rats no significant MFS was observed. These findings indicate that Sema3A mRNA downregulation is temporarily correlated with MFS, while GAP-43 upregulation per se is not, and suggest that a loss of Sema3A in the molecular layer of the dentate gyrus could facilitate MFS into this area during epilepsy.

Postnatal Maternal Separation Elevates the Expression of the Postsynaptic Protein Kinase C Substrate RC3, but Not Presynaptic GAP-43, in the Developing Rat Hippocampus

We have shown that exposure of rats to neonatal handling/maternal separation results in mossy fiber axon hypoplasia in field CA3 of the hippocampus. To better understand the molecular basis of this neuroanatomical alteration, the present study examined three developmentally regulated protein kinase C substrate mRNAs that are highly expressed in hippocampal granule cells during mossy fiber outgrowth: GAP-43, a presynaptic substrate implicated in axonal outgrowth, RC3 (neurogranin), a postsynaptic substrate implicated in calmodulin signaling, and MARCKS-like protein (MLP), which binds calmodulin and filamentous actin in neurons and glial cells. mRNA expression was examined by quantitative in situ hybridization in the developing [postnatal day 7 (P7), P13, P21, and P90] hippocampus (CA1, CA3, granule cells) in Long-Evans hooded rats: (1) reared under normal animal facility (AFR) conditions, (2) subjected to brief (15 min/day, HMS15), or (3) subjected to moderate (180 min/day) handling/maternal separation (HMS180) on P2–14. RC3 mRNA expression was consistently elevated in all of the hippocampal cell fields in HMS180 rats relative to HMS15 and/or AFR rats over postnatal development, but did not differ from HMS15 rats in adulthood. In contrast, neither GAP-43 mRNA nor MLP mRNA expression differed among AFR, HMS15, or HMS180 rats at any postnatal time point. Elevations in RC3 expression would be predicted to perturb calcium-calmodulin signaling that may, in turn, impair the formation and/or maintenance of mossy fiber-CA3 synapses during postnatal development.

Regulation of GAP-43 expression by chronic desipramine treatment in rat cultured hippocampal cells

Background The importance of molecular and cellular changes in hippocampus in major depression and in the mechanism of action of antidepressants has become increasingly clear. Identification of novel targets for antidepressants in hippocampus is important to understanding their therapeutic effects. Methods We used cDNA microarray to measure the expression patterns of multiple genes in primary cultured rat hippocampal cells. In situ hybridization and Northern and immunoblotting analysis were used to determine brain regional distribution and mRNA and protein levels of target genes. Results After comparing hybridized signals between control and desipramine treated groups, we found that chronic treatment with desipramine increased the expression of six genes and decreased the expression of two genes. One of the upregulated genes is growth associated protein GAP-43. In situ hybridization revealed that desipramine increased GAP-43 gene expression in dentate gyrus but not other brain regions. Northern and immunoblotting analysis revealed that desipramine increased GAP-43 mRNA and protein levels. GAP-43 expression is also increased by another antidepressant, tranylcypromine, but not by lithium or haloperidol. Conclusions Because GAP-43 regulates growth of axons and modulates the formation of new connections, our findings suggest that desipramine may have an effect on neuronal plasticity in the central nervous system.

Voluntary exercise induces a BDNF-mediated mechanism that promotes neuroplasticity.

We have investigated potential mechanisms by which exercise can promote changes in neuronal plasticity via modulation of neurotrophins. Rodents were exposed to voluntary wheel running for 3 or 7 days, and their lumbar spinal cord and soleus muscle were assessed for changes in brain-derived neurotrophic factor (BDNF), its signal transduction receptor (trkB), and downstream effectors for the action of BDNF on synaptic plasticity. Exercise increased the expression of BDNF and its receptor, synapsin I (mRNA and phosphorylated protein), growth-associated protein (GAP-43) mRNA, and cyclic AMP response element-binding (CREB) mRNA in the lumbar spinal cord. Synapsin I, a synaptic mediator for the action of BDNF on neurotransmitter release, increased in proportion to GAP-43 and trkB mRNA levels. CREB mRNA levels increased in proportion to BDNF mRNA levels. In separate experiments, the soleus muscle was paralyzed unilaterally via intramuscular botulinum toxin type A (BTX-A) injection to determine the effects of reducing the neuromechanical output of a single muscle on the neurotrophin response to motor activity. In sedentary BTX-A-treated rats, BDNF and synapsin I mRNAs were reduced below control levels in the spinal cord and soleus muscle. Exercise did not change the BDNF mRNA levels in the spinal cord of BTX-A-treated rats but further reduced the BDNF mRNA levels in the paralyzed soleus relative to the levels in sedentary BTX-A-treated rats. Exercise also restored synapsin I to near control levels in the spinal cord. These results indicate that basal levels of neuromuscular activity are required to maintain normal levels of BDNF in the neuromuscular system and the potential for neuroplasticity.

Nerve growth factor controls GAP-43 mRNA stability via the phosphoprotein ARPP-19.

The membrane phosphoprotein GAP-43 is involved in axon growth and synaptic plasticity. In PC12 pheochromocytoma cells, induction of a neuronal phenotype by nerve growth factor (NGF) is accompanied by a marked increase in GAP-43 levels. NGF regulates GAP-43 expression by altering the half-life of its mRNA. We report here that the phosphoprotein ARPP-19 mediates this regulation. In an NGF-dependent manner, ARPP-19 bound to a region in the 3' end of GAP-43 mRNA previously found to be important for regulating the half-life of the mRNA. Overexpression of wild-type ARPP-19 in PC12 cells increased the NGF-dependent expression of a reporter construct linked to the critical 3' region of GAP-43 mRNA. Mutation of serine 104, the site of phosphorylation by protein kinase A in ARPP-19, to either alanine or aspartate abolished this regulation in PC12 cells. These findings demonstrate that ARPP-19 is an important link between NGF signaling and post-transcriptional control of neuronal gene expression.

Expression of MARCKS mRNA in lateral geniculate nucleus and visual cortex of normal and monocularly deprived macaque monkeys.

We performed a nonradioactive in situ hybridization histochemistry (ISH) study of the lateral geniculate nucleus (LGN) and the primary visual area (area 17) of the macaque monkey to investigate mRNA expression of the myristoylated alanine-rich C-kinase substrate (MARCKS), a major protein kinase C (PKC) substrate. In the LGN, intense hybridization signals were observed in both magnocellular neurons (layers 1 and 2) and parvocellular neurons (layers 3 to 6). Double labeling using ISH and immunofluorescence revealed that MARCKS mRNA was coexpressed with the alpha-subunit of type II calcium/calmodulin-dependent protein kinase, indicating that MARCKS mRNA is also expressed in koniocellular neurons in the LGN. GABA-immunoreactive neurons in the LGN did not contain MARCKS mRNA, indicating that MARCKS mRNA is not expressed in inhibitory interneurons. The signals were generally weak in area 17, and intense signals were restricted to large neurons in layers IVB, V, and VI. GABA-immunoreactive neurons in layers II-VI of area 17 did not contain MARCKS mRNA. Double-label ISH revealed that MARCKS mRNA was coexpressed with mRNA of GAP-43, another PKC substrate, in neurons of both the LGN and area 17. To determine whether the expression of MARCKS mRNA is regulated by retinal activity, we performed ISH in the LGN and area 17 of monkeys deprived of monocular visual input by tetrodotoxin. After monocular deprivation for 5 to 30 days, MARCKS mRNA was down-regulated in the LGN, but not in area 17. These results suggest that MARCKS mediates the activity-dependent changes in the excitatory relay neurons in the LGN.

Transcriptional Upregulation of SCG10 and CAP-23 Is Correlated with Regeneration of the Axons of Peripheral and Central Neurons in Vivo

We have compared SCG10 and CAP-23 expression with that of GAP-43 during axonal regeneration in the peripheral and central nervous systems (PNS, CNS) of adult rats. SCG10, CAP-23, and GAP-43 mRNAs were strongly upregulated by motor and dorsal root ganglion (DRG) neurons following sciatic nerve crush, but not after dorsal rhizotomy. When the sciatic nerve was cut and ligated to prevent reinnervation of targets, expression of all three mRNAs was prolonged. Neurons in the thalamic reticular nucleus and deep cerebellar nuclei transiently upregulated these mRNAs after axotomy, and showed prolonged upregulation of all three molecules when regenerating axons into peripheral nerve grafts inserted into the thalamus of cerebellum. Neurons in the dorsal thalamus and cerebellar cortex showed poor regenerative capacity and most did not upregulate any of these mRNAs. Thus, in both PNS and CNS neurons, the transcription of SCG10, CAP-23, and GAP-43 mRNAs is coregulated following axotomy and during regeneration. Signals from living peripheral nerve appear to maintain expression of all three mRNAs in regenerating neurons, and in PNS neurons downregulation correlates with target reinnervation. Thus, SCG10 and CAP-23, as well as GAP-43, are likely to be important neuronal determinants of regenerative ability.

Developmental Down-Regulation of GAP-43 Expression and Timing of Target Contact in Rat Corticospinal Neurons

During early nervous system development axons grow toward the target tissue that they will innervate. As axons invade target tissue, growth slows and ceases. Neurons express high levels of the growth-associated protein GAP-43 during developmental axon growth, declining with maturation. It has been suggested that target contact provides a signal which down-regulates GAP-43 expression. To study this issue in more detail, we used in situ hybridization to quantify relative changes in GAP-43 mRNA in corticospinal tract neurons identified by Fast Blue retrograde labeling. We also used anterograde transport of biotinylated dextran amine to study the invasion of target by corticospinal axons. We find that GAP-43 mRNA is high during the first postnatal week and then declines in two phases. Approximately half of the initial level of GAP-43 expression in corticospinal neurons is lost by P12; then expression remains at a plateau until P21. Between P21 and P28, GAP-43 expression again declines by half and then remains steady at the adult level (one fourth of initial level). Corticospinal axons initially invade spinal gray matter during the first 2 postnatal weeks, in a rostrocaudal gradient. Varicosities suggestive of terminal boutons become numerous during the third and fourth week, and the morphology of corticospinal axon terminals achieves the mature form at the end of the fourth week. These data suggest that the first phase of down-regulation of GAP-43 in corticospinal neurons is coincident with initial target contact and that the second phase is coincident with final maturation of terminal arborization.

Rho signaling pathway targeted to promote spinal cord repair.

The Rho signaling pathway regulates the cytoskeleton and motility and plays an important role in neuronal growth inhibition. Here we demonstrate that inactivation of Rho or its downstream target Rho-associated kinase (ROK) stimulated neurite growth in primary cells of cortical neurons plated on myelin or chondroitin sulfate proteoglycan substrates. Furthermore, treatment either with C3 transferase (C3) to inactivate Rho or with Y27632 to inhibit ROK was sufficient to stimulate axon regeneration and recovery of hindlimb function after spinal cord injury (SCI) in adult mice. Injured mice were treated with a single injection of Rho or Rho-associated kinase inhibitors delivered in a protein adhesive at the lesion site. Treated animals showed long-distance regeneration of anterogradely labeled corticospinal axons and increased levels of GAP-43 mRNA in the motor cortex. Behaviorally, inactivation of Rho pathway induced rapid recovery of locomotion and progressive recuperation of forelimb-hindlimb coordination. These findings provide evidence that the Rho signaling pathway is a potential target for therapeutic interventions after spinal cord injury.

Rho Signaling Pathway Targeted to Promote Spinal Cord Repair

The Rho signaling pathway regulates the cytoskeleton and motility and plays an important role in neuronal growth inhibition. Here we demonstrate that inactivation of Rho or its downstream target Rho-associated kinase (ROK) stimulated neurite growth in primary cells of cortical neurons plated on myelin or chondroitin sulfate proteoglycan substrates. Furthermore, treatment either with C3 transferase (C3) to inactivate Rho or with Y27632 to inhibit ROK was sufficient to stimulate axon regeneration and recovery of hindlimb function after spinal cord injury (SCI) in adult mice. Injured mice were treated with a single injection of Rho or Rho-associated kinase inhibitors delivered in a protein adhesive at the lesion site. Treated animals showed long-distance regeneration of anterogradely labeled corticospinal axons and increased levels of GAP-43 mRNA in the motor cortex. Behaviorally, inactivation of Rho pathway induced rapid recovery of locomotion and progressive recuperation of forelimb-hindlimb coordination. These findings provide evidence that the Rho signaling pathway is a potential target for therapeutic interventions after spinal cord injury.

Cytokines and neurotrophic factors fail to affect Nogo-A mRNA expression in differentiated human neurones: implications for inflammation-related axonal regeneration in the central nervous system.

Nogo is a novel myelin-associated inhibitor of neurite outgrowth which regulates stable neuronal connections during axonal regeneration following injury in the adult mammalian central nervous system (CNS). Because cytokines and neurotrophic factors play a key role in inflammation-related axonal regeneration, we investigated: (i) the constitutive expression of Nogo and the Nogo receptor (NgR) mRNA in human neural cell lines; (ii) Nogo and NgR mRNA levels in the NTera2 human teratocarcinoma cell line during retinoic acid (RA)-induced neuronal differentiation; and (iii) their regulation in NTera2-derived differentiated neurones (NTera2-N) after exposure to a battery of cytokines and growth factors potentially produced by activated glial cells at post-traumatic inflammatory lesions in the CNS. By reverse transcriptase-polymerase chain reaction analysis, the constitutive expression of Nogo-A, the longest isoform of three distinct Nogo transcripts and NgR mRNA was identified in a wide variety of human neural and non-neural cell lines. By Northern blot analysis, the levels of Nogo-A mRNA were elevated markedly in NTera2 cells following RA-induced neuronal differentiation, accompanied by an increased expression of the neurite growth-associated protein GAP-43 mRNA. In contrast, Nogo-A, Nogo-B, NgR and GAP-43 mRNA levels were unaltered in NTera2-N cells by exposure to basic fibroblast growth factor, brain-derived neurotrophic factor, glia-derived neurotrophic factor, tumour necrosis factor-alpha, interleukin-1beta, dibutyryl cyclic AMP or phorbol 12-myristate 13-acetate. These results indicate that both Nogo-A and NgR mRNA are coexpressed in various human cell types, including differentiated neurones, where their expression is unaffected by exposure to a panel of cytokines and neurotrophic factors which might be involved in inflammation-related axonal regeneration in the CNS.

Role of HuD and other RNA-binding proteins in neural development and plasticity.

Transcription factors have traditionally been viewed as the main determinants of gene expression. Yet, in recent years it has become apparent that RNA-binding proteins also play a critical role in determining the levels of expression of a large number of genes. Once mRNAs are transcribed, RNA-binding proteins can control all subsequent steps in their function, from alternative splicing and translation to mRNA transport and stability. In the nervous system, a large number of genes are regulated post-transcriptionally via the interaction of their mRNAs with specific RNA-binding proteins. This type of regulation is particularly important in the control of the temporal and spatial pattern of gene expression during neural development. This review will discuss the function of the embryonic lethal abnormal vision (ELAV)/Hu family of nervous system-specific RNA-binding proteins, with a special emphasis on HuD, a member of this family that controls GAP-43 mRNA stability and expression. In addition, we will present recent findings on other neural RNA-binding proteins: the ribonucleoprotein K homology (KH)-domain proteins, Fragile X mental retardation protein (FMRP), quakinguiable protein (QKI), and Nova-1. Together with the ELAV/Hu family, these proteins are essential for proper neural development and in some cases for plasticity in the mature brain. The biological significance of these proteins is evident not only by their evolutionary conservation but also by the magnitude of problems arising from autoimmune reactions against them or from mutations affecting their expression or function.