An in vitro assay to study induction of the regenerative state in sensory neurons.

After injury, peripheral neurons activate a pro-regenerative program that facilitates axon regeneration. While many regeneration-associated genes have been identified, the mechanism by which injury activates this program is less well understood. Furthermore, identifying pharmacological methods to induce a pro-regenerative state could lead to novel treatments to repair the injured nervous system. Therefore, we have developed an in vitro assay to study induction of the pro-regenerative state following injury or pharmacological treatment. First, we took advantage of the observation that dissociating and culturing sensory neurons from dorsal root ganglia activates a pro-regenerative program. We show that cultured neurons activate transcription factors and upregulate regeneration-associated genes common to the pro-regenerative program within the first hours after dissection. In a paradigm similar to pre-conditioning, neurons injured by dissociation display enhanced neurite outgrowth when replated as early as 12h after being removed from the animal. Furthermore, stimulation of the pro-regenerative state improves growth on inhibitory substrates and requires DLK/JNK signaling, both hallmarks of the pro-regeneration response in vivo. Finally, we modified this assay in order to identify new methods to activate the pro-regenerative state in an effort to mimic the pre-conditioning effect. We report that after several days in culture, neurons down-regulate many molecular hallmarks of injury and no longer display enhanced neurite outgrowth after replating. Hence, these neurons are functionally naïve and are a useful tool for identifying methods to induce the pro-regenerative state. We show that both injury and pre-treatment with forskolin reactivate the pro-regenerative state in this paradigm. Hence, this assay is useful for identifying pharmacological agents that induce the pro-regenerative state in the absence of injury.

Loss of Nkx3.1 leads to the activation of discrete downstream target genes during prostate tumorigenesis.

The expression of NKX3.1, a transcriptional regulator and tumor suppressor gene in prostate cancer, is downregulated during early stages of prostate tumorigenesis. However, little is known of the alterations in gene expression that occur as a result of this event. We combined laser capture microdissection and gene expression profiling to analyse the molecular consequences of Nkx3.1 loss during prostate cancer initiation using Nkx3.1-deficient mice. This analysis identified a cohort of genes (loss-of-Nkx3.1 signature) that are aberrantly overexpressed during loss-of-Nkx3.1-driven tumor initiation. We studied the expression of these genes in independent loss-of-Pten and c-myc overexpression prostate adenocarcinoma mouse models. Nkx3.1 expression is lost in prostate epithelial proliferation in both of these mouse models. However, Nkx3.1 loss is an early event of tumor development in the loss-of-Pten model, whereas it occurs at later stages in c-myc transgenic mice. A number of genes of the loss-of-Nkx3.1 signature, such as clusterin and quiescin Q6, are highly expressed in prostatic hyperplasia and intraepithelial neoplasia (PIN) lesions that also lack Nkx3.1 in the Pten-deficient prostate, but not in similar lesions in the c-myc transgenic model. Meta-analysis of multiple prostate cancer gene expression data sets, including those from loss-of-Nkx3.1, loss-of-Pten, c-myc overexpression and constitutively active Akt prostate cancer models, further confirmed that genes associated with the loss-of-Nkx3.1 signature integrate with PTEN-AKT signaling pathways, but do not overlap with molecular changes associated with the c-myc signaling pathway. In human prostate tissue samples, loss of NKX3.1 expression and corresponding clusterin overexpression are co-localized at sites of prostatic inflammatory atrophy, a possible very early stage of human prostate tumorigenesis. Collectively, these results suggest that the molecular consequences of NKX3.1 loss depend on the epithelial proliferative stage at which its expression is lost, and that alterations in the PTEN-AKT-NKX3.1 axis are important for prostate cancer initiation.

Analysis of Ret knockin mice reveals a critical role for IKKs, but not PI 3-K, in neurotrophic factor-induced survival of sympathetic neurons.

We analyzed the survival responses and downstream signaling elicited by GDNF on sympathetic neurons from different Ret knockin mice. Lack of tyrosine 1062, a multidocking site in Ret, completely prevented GDNF-mediated survival. Importantly, lack of tyrosine 981, although abrogating Akt phosphorylation, had no effect on neuronal survival, indicating that the PI 3-K/Akt pathway is not necessary for survival of sympathetic neurons. In contrast, silencing of B-Raf completely prevented not only GDNF-mediated but also NGF-mediated cell survival, independently of MEK-1/2. We identified IKKs as the main effectors of the protective effects of B-Raf. First, B-Raf interacted with and activated IKKs. Second, knockdown of IKKs reversed the protection afforded by a constitutively active form of B-Raf. Third, knockdown of IKKs prevented both NGF- and GDNF-mediated survival. In conclusion, our data delineate a novel survival pathway for sympathetic neurons linking B-Raf to IKKs, independently of both PI 3-K and MEK-1/2 pathways.

The immediate early gene early growth response gene 3 mediates adaptation to stress and novelty.

Stress and exploration of novel environments induce neural expression of immediate early gene transcription factors (IEG-TFs). However, as yet no IEG-TF has been shown to be required for the normal biological or behavioral responses to these stimuli. Here we show that mice deficient for the IEG-TF early growth response gene (Egr) 3, display accentuated behavioral responses to the mild stress of handling paralleled by increased release of the stress hormone corticosterone. Egr3-/- mice also display abnormal responses to novelty, including heightened reactivity to novel environments and failure to habituate to social cues or startling acoustic stimuli. In a Y-maze spontaneous alternation task, they perform fewer sequential arm entries than controls, suggesting defects in immediate memory. Because stress and novelty stimulate hippocampal long-term depression (LTD), and because abnormalities in habituation to novelty and Y-maze performance have been associated with LTD deficits, we examined this form of synaptic plasticity in Egr3-/- mice. We found that Egr3-/- mice fail to establish hippocampal LTD in response to low frequency stimulation and exhibit dysfunction of an ifenprodil-sensitive (NR1/NR2B) N-methyl-d-aspartate receptor subclass. Long term potentiation induction was not altered. The NR2B-dependent dysfunction does not result from transcriptional regulation of this subunit by Egr3, because NR2B mRNA levels did not differ in the hippocampi of Egr3-/- and control mice. These findings are the first demonstration of the requirement for an IEG-TF in mediating the response to stress and novelty, and in the establishment of LTD.

Dynamics of neural crest-derived cell migration in the embryonic mouse gut.

Neural crest-derived cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastrointestinal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest-derived cell migration in the embryonic mouse gut in organ culture. Time-lapse imaging revealed that GFP(+) crest-derived cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells and their processes formed a scaffold along which later cells migrated. To examine the effect of population size on migratory behavior, a small number of the most caudal GFP(+) cells were isolated from the remainder of the population. The isolated cells migrated slower than cells in large control populations, suggesting that migratory behavior is influenced by cell number and cell-cell contact. Previous studies have shown that neurons differentiate among the migrating cell population, but it is unclear whether they migrate. The phenotype of migrating cells was examined. Migrating cells expressed the neural crest cell marker, Sox10, but not neuronal markers, indicating that the majority of migratory cells observed did not have a neuronal phenotype.

Neurturin signalling via GFRalpha2 is essential for innervation of glandular but not muscle targets of sacral parasympathetic ganglion neurons.

Neurturin, a member of the glial cell-derived neurotrophic factor familys of ligands, is important for development of many cranial parasympathetic ganglion neurons. We have investigated the sacral component of the parasympathetic nervous system in mice with gene deletions for neurturin or its preferred receptor, GFRalpha2. Disruption of neurturin signalling decreased cholinergic VIP innervation to the mucosa of the reproductive organs, but not to the smooth muscle layers of these organs or to the urinary bladder. Thus, neurturin and its receptor are involved in parasympathetic innervation of a select group of pelvic visceral tissues. In contrast, noradrenergic innervation was not affected by the gene ablations. The epithelium of reproductive organs from knockout animals was atrophied, indicating that cholinergic innervation may be important for the maintenance of normal structure. Cholinergic neurons express GFRalpha2 on their terminals and somata, indicating they can respond to neurotrophic support, and their somata are smaller when neurturin signalling is disrupted. Colocalisation studies showed that many peripheral glia express GFRalpha2 although its role in these cells is yet to be determined. Our results indicate that neurturin, acting through GFRalpha2, is essential for parasympathetic innervation of the mucosae of reproductive organs, as well as for maintenance of a broader group of sacral parasympathetic neurons.

Idiopathic slow transit constipation and megacolon are not associated with neurturin mutations.

Chronic idiopathic slow-transit constipation (ISTC) and idiopathic megacolon (IMC) are early-onset gastrointestinal motility disorders of unknown aetiology. The gene encoding the neurotrophic factor neurturin may be a candidate for these disorders, as neurturin-deficient mice have a similar enteric phenotype. In the present study, we tested this hypothesis. Genomic DNA from 26 cases of chronic idiopathic STC [with a family history of constipation in 15 (58%) and Hirschsprung's disease in two (8%)], and five cases of IMC [two familial (40%)] was screened by direct DNA sequencing using the fluorescent dideoxy terminator method. Results were compared with published sequence data and 24 control DNAs. Our results revealed several previously unreported common sequence polymorphisms, but overall frequencies were comparable between patients and controls. We conclude that mutation of neurturin is not a frequent cause of ISTC or IMC.

GDNF and neurturin are target-derived factors essential for cranial parasympathetic neuron development.

During development, parasympathetic ciliary ganglion neurons arise from the neural crest and establish synaptic contacts on smooth and striate muscle in the eye. The factors that promote the ciliary ganglion pioneer axons to grow toward their targets have yet to be determined. Here, we show that glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) constitute target-derived factors for developing ciliary ganglion neurons. Both GDNF and NRTN are secreted from eye muscle located in the target and trajectory pathway of ciliary ganglion pioneer axons during the period of target innervation. After this period, however, the synthesis of GDNF declines markedly, while that of NRTN is maintained throughout the cell death period. Furthermore, both in vitro and in vivo function-blocking of GDNF at early embryonic ages almost entirely suppresses ciliary axon outgrowth. These results demonstrate that target-derived GDNF is necessary for ciliary ganglion neurons to innervate ciliary muscle in the eye. Since the down-regulation of GDNF in the eye is accompanied by down-regulation of GFRalpha1 and Ret, but not of GFRalpha2, in innervating ciliary ganglion neurons, the results also suggest that target-derived GDNF regulates the expression of its high-affinity coreceptors.

RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons.

Sympathetic axons use blood vessels as an intermediate path to reach their final target tissues. The initial contact between differentiating sympathetic neurons and blood vessels occurs following the primary sympathetic chain formation, where precursors of sympathetic neurons migrate and project axons along or toward blood vessels. We demonstrate that, in Ret-deficient mice, neuronal precursors throughout the entire sympathetic nervous system fail to migrate and project axons properly. These primary deficits lead to mis-routing of sympathetic nerve trunks and accelerated cell death of sympathetic neurons later in development. Artemin is expressed in blood vessels during periods of early sympathetic differentiation, and can promote and attract axonal growth of the sympathetic ganglion in vitro. This analysis identifies RET and artemin as central regulators of early sympathetic innervation.

Frequent and early loss of the EGR1 corepressor NAB2 in human prostate carcinoma.

The transcription factor EGR1 is frequently overexpressed in human prostate cancer and regulates the expression of several genes important for tumor progression. In addition, mice lacking the Egr1 gene show a defect in prostate tumorigenesis. NAB2 is a novel corepressor molecule that modulates EGR1 activity and is induced by the same stimuli that induce EGR1. The human NAB2 gene has been localized to 12q13.3-14.1, within a chromosomal region that is thought to harbor a prostate tumor suppressor. We have examined the expression of NAB2 in human prostate carcinoma specimens. We show here that NAB2 protein expression is lost in a majority of primary prostate carcinoma specimens, including many samples that have high EGR1 levels. This loss occurs early in the tumorigenic process and is sustained, as it is seen in precursor prostatic intraepithelial neoplasia lesions as well as in metastases. Furthermore, loss of NAB2 did not correlate with the tumor grade or stage. Our findings suggest that high levels of EGR1 coupled with low levels of NAB2 can result in high, unrestrained EGR1 transcriptional activity in human prostate cancers.

Expression profiling reveals hepsin overexpression in prostate cancer.

Prostate cancer is the most commonly diagnosed noncutaneous cancer in men. Despite this fact, many of the genetic changes that coincide with prostate cancer progression remain enigmatic. We have addressed this problem by characterizing the expression profiles of several benign and malignant human prostate samples, and we have identified several genes that are differentially expressed between benign and malignant glands. One gene that was overexpressed encodes the serine protease hepsin. We used an independent sample set to confirm that hepsin is overexpressed in prostate tumors, and in situ hybridization demonstrates that hepsin is specifically overexpressed in the carcinoma cells themselves. These facts, together with the molecular properties of hepsin, make it an ideal target for prostate cancer therapy.

The transcription factor Egr3 modulates sensory axon-myotube interactions during muscle spindle morphogenesis.

The Egr family of zinc-finger transcription factors, consisting of Egr1, Egr2, Egr3, and Egr4, are involved in cellular growth and differentiation. Adult Egr3-deficient mice are ataxic and lack muscle spindle proprioceptors that normally develop at the sites of Ia afferent-myotube contacts during embryogenesis. To resolve whether spindles form and then degenerate, or whether they never form in the absence of Egr3, we examined the spatiotemporal expression of Egr3 relative to spindle development. In wild type mice, Egr3 was expressed in developing myotubes shortly after they were innervated by Ia afferents and its expression was controlled by innervation because it dissipated following nerve transection. In Egr3-deficient mice, myotubes received Ia afferent innervation and assembled normally into spindles during embryogenesis. However, newborn Egr3-deficient spindles had few internal myonuclei in intrafusal fibers and thin capsules. Moreover, slow-developmental myosin heavy chain was not induced in embryonic Egr3-deficient spindles suggesting that impairments in differentiation were present before they could be detected morphologically. After birth, sensory and motor innervation withdrew from the Egr3-deficient spindles, and the spindles disassembled. In spite of the spindle disassembly and retraction of afferents from muscles, the cell bodies of proprioceptive neurons within dorsal root ganglia were retained. We conclude that Egr3 has an essential role in regulating genes required for the transformation of undifferentiated myotubes into intrafusal fibers, and hence for the phenotypic differentiation of spindles.

EGR2 mutations in inherited neuropathies dominant-negatively inhibit myelin gene expression.

The identification of EGR2 mutations in patients with neuropathies and the phenotype Egr2/Krox20(-/-) have demonstrated that the Egr2 transcription factor is critical for peripheral nerve myelination. However, the mechanism by which these mutations cause disease remains unclear, as most patients present with disease in the heterozygous state, whereas Egr2(+/-) mice are phenotypically normal. To understand the effect of aberrant Egr2 activity on Schwann cell gene expression, we performed microarray expression profiling to identify genes regulated by Egr2 in Schwann cells. These include genes encoding myelin proteins and enzymes required for synthesis of normal myelin lipids. Using these newly identified targets, we have shown that neuropathy-associated EGR2 mutants dominant-negatively inhibit wild-type Egr2-mediated expression of essential myelin genes to levels sufficiently low to result in the abnormal myelination observed in these patients.

Identification of genes induced in peripheral nerve after injury. Expression profiling and novel gene discovery.

Peripheral nerve injury results in axonal degeneration and in phenotypic changes of the surrounding Schwann cells, whose presence is critical for nerve regeneration. To identify genes induced after nerve injury in Schwann cells, we developed a strategy that included differential screening of a subtractive library enriched for cDNAs expressed in injured nerve, sequence analysis, and expression profiling. By using real time quantitative reverse transcriptase-polymerase chain reaction, we found that injury-induced genes could be categorized into four temporal expression patterns. Among the clones we identified were a number that were homologous only to expressed sequence tags in the data base. These were stratified based on their expression profile, presence of identifiable sequence motifs, homologies to other proteins, and evolutionary conservation. We chose one representative gene, nin283, to analyze in detail. The nin283 gene encodes a 227-residue protein containing both a zinc finger and a RING finger motif. nin283 is highly expressed in the central nervous system, particularly in the developing cortical plate in embryos. It is also expressed in peripheral ganglia and is induced by nerve growth factor in PC12 cells. Subcellular localization analysis demonstrated that Nin283 is located in the endosome/lysosome compartment, suggesting that it may participate in ubiquitin-mediated protein modification.

The transcriptional corepressor NAB2 blocks Egr-1-mediated growth factor activation and angiogenesis.

Effective tissue repair results from a rapid, temporally orchestrated series of events. At the site of local tissue injury, the production of many growth factors and cytokines is, in part, stimulated by the early growth response transcription factors such as Egr-1. Egr-1 protein binds to a family of corepressor proteins called NAB which function to block or limit Egr-1 trans-activation of cognate target genes. NAB2 blocks Egr-1 activation of the tissue factor (TF) promoter, Egr-1 stimulated production of PDGF-AB, HGF, TGFbeta(1), and VEGF and the endogenous expression of PDGF-AB and TGFbeta(1). Expression of a wild-type NAB2 but not a dominant negative NAB2 mutant abrogates Egr-1 driven TF promoter activity and tubule formation in an in vitro model of angiogenesis. These findings may have importance in any tissue that is subject to scarring after acute or chronic injury.

Epitope-tagged recombinant AAV vectors for expressing neurturin and its receptor in retinal cells.

PURPOSE: Neurturin (NTN) is a potent neuronal survival factor in the central and peripheral nervous systems. We previously described altered expression of mRNAs for NTN and one of its receptor components, GFRa-2 in degenerative retinas of rd/rd mice. Towards assessing the potential for transfer of these genes to counteract retinal degeneration, we examined recombinant adeno-associated virus (rAAV) constructs for expression of NTN and GFRa-2 transgenes in retinal cells in vitro and for the effect of transgene expression on retinal function following intraocular delivery in rd/rd mice. METHODS: The rAAV constructs incorporated epitope tags to facilitate discrimination between transgenic and endogenous expression. Expression of murine NTN was driven by a CMV promoter and a partial murine opsin promoter was used to drive expression of human GFRa-2. rAAV preparations were used to infect mouse retinal cell cultures and for intraocular injection of predegenerative rd/rd mice. Endogenous and transgene expression was analyzed by immunofluorescence. Photoreceptor function in treated mice was assessed by electroretinography. RESULTS: Both vectors delivered and expressed their transgenes in vitro and in vivo. Differential targeting was achieved in vivo through the use of alternative promoters. Under the conditions examined, no functional rescue of rd photoreceptors was observed. CONCLUSIONS: Therapeutic treatment of the rd model of retinal degeneration does not appear to be effected by simple modulation of the expression of NTN or GFRa-2, and may therefore depend on additional synergistic factors. Our AAV constructs will facilitate the development of combinatorial approaches to the treatment of central and peripheral neurodegenerations.

Differential expression of Fas ligand in Th1 and Th2 cells is regulated by early growth response gene and NF-AT family members.

Inducible expression of Fas ligand (CD95 ligand) by activated T cells and the resulting apoptosis of CD95-bearing cells is a critical component of peripheral T cell homeostasis and cytotoxic effector mechanisms. Transcriptional control of the expression of Fas ligand has been attributed to a number of factors, including early growth response gene 2 (Egr2), Egr3, Sp1, and NF-AT, although a direct contribution of NF-AT is controversial. The present study confirms a role for Egr factors and indicates that NF-AT is essential for optimal expression of murine Fas ligand through a direct interaction with an NF-AT consensus element. The role of these factors was further defined by studying the differential expression of Fas ligand in Th1 and Th2 lines derived from DO11.10 TCR transgenic mice. EMSA analyses of a composite Egr/NF-AT site showed recruitment of Sp1 to this site in Th2 cells, but not in Th1 cells. Furthermore, gel-shift analyses demonstrated the binding of Egr1, 2, and 3 in Th2 cells and Egr1 and 2, but not Egr3 in Th1 cells at a known Egr site. Northern analysis corroborated the lack of Egr3 in Th1 cells. Differential usage of these transcription factors by Th1 and Th2 cells suggests a potential mechanism underlying the differential expression of Fas ligand by distinct T cell lineages.

The transcription factor early growth-response factor 1 modulates tumor necrosis factor-alpha, immunoglobulin E, and airway responsiveness in mice.

Early growth-response factor 1 (Egr-1) is a sequence-specific transcription factor that plays a regulatory role in the expression of many genes important in inflammation, cell growth, apoptosis, and the pathogenesis of disease. In vitro studies suggest that Egr-1 is capable of regulating the expression of tumor necrosis factor-alpha (TNF-alpha) and other genes involved in airway inflammation and reactivity following allergen stimulation. On the basis of these data, we hypothesized that in the absence of Egr-1, the TNF-alpha response and subsequent downstream inflammatory events that usually follow allergen challenge would be diminished. To test our hypothesis Egr-1 knock-out (KO) mice were examined in an ovalbumin (OVA)-induced model of airway inflammation and reactivity, and compared with identically treated wild-type (WT) control mice. In response to OVA sensitization and airway challenge, KO mice had diminished TNF-alpha mRNA and protein in the lungs and mast cells compared with WT mice. Interestingly, the KO mice had elevated IgE levels at baseline and after allergen challenge compared with WT mice. Furthermore, the airways of KO mice were hyporesponsive to methacholine challenge at baseline and after allergen challenge. These data indicate that Egr-1 modulates TNF-alpha, IgE, and airway responsiveness in mice.

Quantitative amplification of genomic DNA from histological tissue sections after staining with nuclear dyes and laser capture microdissection.

Laser capture microdissection (LCM) allows the selective sampling of tissue from histological sections. A prerequisite for this technique is the availability of histological dyes that do not interfere with downstream analysis of the sampled genetic material. We have examined the effect of four histological nuclear dyes (methyl green, hematoxylin, toluidine blue O, azure B) on TaqMan polymerase chain reaction amplification of beta-actin genomic DNA prepared from fixed and frozen tissue. Tissue sampled from the histological sections by manual dissection was compared with tissue sampled by LCM. As previously reported, when manually dissected tissue sections were analyzed, polymerase chain reaction amplification of DNA after hematoxylin staining was inferior to that after staining with the other dyes. In contrast, when tissue sampled by LCM was examined, DNA recovery after hematoxylin staining was equivalent to the recovery after methyl green staining. We conclude that DNA recovery from LCM-sampled tissue is independent of the histological stain chosen to highlight nuclear detail.