Activation Of Non-neuronal Cells Research Articles

Micellar Choline-Acetyltransferase Complexes Exhibit Ultra-Boosted Catalytic Rate for Acetylcholine Synthesis-Mechanistic Insights for Development of Acetylcholine-Enhancing Micellar Nanotherapeutics.

Choline-acetyltransferase (ChAT) is the key cholinergic enzyme responsible for the biosynthesis of acetylcholine (ACh), a crucial signaling molecule with both canonical neurotransmitter function and auto- and paracrine signaling activity in non-neuronal cells, such as lymphocytes and astroglia. Cholinergic dysfunction is linked to both neurodegenerative and inflammatory diseases. In this study, we investigated a serendipitous observation, namely that the catalytic rate of human recombinant ChAT (rhChAT) protein greatly differed in buffered solution in the presence and absence of Triton X-100 (TX100). At a single concentration of 0.05% (v/v), TX100 boosted the specific activity of rhChAT by 4-fold. Dose-response analysis within a TX100 concentration range of 0.8% to 0.008% (accounting for 13.7 mM to 0.013 mM) resulted in an S-shaped response curve, indicative of an over 10-fold boost in the catalytic rate of rhChAT. This dramatic boost was unlikely due to a mere structural stabilization since it remained even after the addition of 1.0 mg/mL gelatin to the ChAT solution as a protein stabilizer. Furthermore, we found that the catalytic function of the ACh-degrading enzyme, AChE, was unaffected by TX100, underscoring the specificity of the effect for ChAT. Examination of the dose-response curve in relation to the critical micelle concentration (CMC) of TX100 revealed that a boost in ChAT activity occurred when the TX100 concentration passed its CMC, indicating that formation of micelle-ChAT complexes was crucial. We challenged this hypothesis by repeating the experiment on Tween 20 (TW20), another non-ionic surfactant with ~3-fold lower CMC compared to TX100 (0.06 vs. 0.2 mM). The analysis confirmed that micelle formation is crucial for ultra-boosting the activity of ChAT. In silico molecular dynamic simulation supported the notion of ChAT-micelle complex formation. We hypothesize that TX100 or TW20 micelles, by mimicking cell-membrane microenvironments, facilitate ChAT in accessing its full catalytic potential by fine-tuning its structural stabilization and/or enhancing its substrate accessibility. These insights are expected to facilitate research toward the development of new cholinergic-enhancing therapeutics through the formulation of micelle-embedded ChAT nanoparticles.

Role of optogenetics in pain management

Optogenetics is a biological technique that involves the use of light to control genetically modified neurons to express the channels of light-sensitive Ions. Optogenetics can also be referred to as Optical monitoring of neuronal activity in non-neuronal cells, and control of biochemical pathways. Optogenetics is less invasive than electrical stimulation response time in optogenetics is comparatively faster than other treatments. Chronic pain affects one-third of the population, and current treatments cause limited relief and serious side effects. An alternative approach to pain reduction would be the direct modulation of somatosensory pathways using optogenetics. Optogenetic treatment can be a very effective way to relieve chronic pain while avoiding conventional pain medicine's side effects. The use of optogenetics in pain management has greatly accelerated over the last decade. This review discusses the versatility of different optogenetic tools and its effective applications in the field of medicine.

Sympathetic Input to Multiple Cell Types in Mouse and Human Colon Produces Region-Specific Responses

The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells). We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single-cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function. Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of α-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings. Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these 2 regions.

Immunofluorescence reveals neuron-specific promoter activity in non-neuronal cells.

Immunofluorescence reveals neuron-specific promoter activity in non-neuronal cells.

RNA-seq of spinal cord from nerve-injured rats after spinal cord stimulation.

Spinal cord stimulation has become an important modality in pain treatment especially for neuropathic pain conditions refractory to pharmacotherapy. However, the molecular control of inhibitory and excitatory mechanisms observed after spinal cord stimulation are poorly understood. Here, we used RNA-seq to identify differences in the expression of genes and gene networks in spinal cord tissue from nerve-injured rats with and without repetitive conventional spinal cord stimulation treatment. Five weeks after chronic constrictive injury to the left sciatic nerve, male and female rats were randomized to receive repetitive spinal cord stimulation or no treatment. Rats receiving spinal cord stimulation underwent epidural placement of a miniature stimulating electrode and received seven sessions of spinal cord stimulation (50 Hz, 80% motor threshold, 0.2 ms, constant current bipolar stimulation, 120 min/session) over four consecutive days. Within 2 h after the last spinal cord stimulation treatment, the L4-L6 spinal segments ipsilateral to the side of nerve injury were harvested and used to generate libraries for RNA-seq. Our RNA-seq data suggest further increases of many existing upregulated immune responses in chronic constrictive injury rats after repetitive spinal cord stimulation, including transcription of cell surface receptors and activation of non-neuronal cells. We also demonstrate that repetitive spinal cord stimulation represses transcription of several key synaptic signaling genes that encode scaffold proteins in the post-synaptic density. Our transcriptional studies suggest a potential relationship between specific genes and the therapeutic effects observed in patients undergoing conventional spinal cord stimulation after nerve injury. Furthermore, our results may help identify new therapeutic targets for improving the efficacy of conventional spinal cord stimulation and other chronic pain treatments.

Engineered HSV vector achieves safe long-term transgene expression in the central nervous system

Previously we reported a new series of highly defective herpes simplex virus type 1 (HSV-1) vectors that were functionally devoid of all viral immediately early (IE) genes, resulting in virtual absence of viral gene expression. Nevertheless, a reporter gene cassette inserted into the vector flanked by boundary elements from the viral latency locus showed high, persistent reporter gene activity in non-neuronal cells while an independent expression cassette inserted into a deleted ICP4 locus remained almost silent. In contrast to non-neuronal cells, we show here that the ICP4 locus cassette permitted robust reporter gene expression in a diversity of neurons following stereotactic injection of different rat brain regions; transgene expression in the hippocampus lasted up to 6 months and was essentially restricted to neurons. No evidence of neuronal cell toxicity or induction of inflammatory cell infiltrates was observed. An independent reporter gene cassette located in an intergenic region remained silent, indicating that the transgene promoter and/or insertion site are critical for sustained expression. These findings suggest the suitability of this vector for therapeutic intervention into diseases of the central nervous system that require the expression of large and/or multiple therapeutic transgenes.

Minimal NF-κB activity in neurons

Nuclear factor-kappa B (NF-κB) is a ubiquitous transcription factor that regulates immune and cell-survival signaling pathways. NF-κB has been reported to be present in neurons wherein it reportedly responds to immune and toxic stimuli, glutamate, and synaptic activity. However, because the brain contains many cell types, assays specifically measuring neuronal NF-κB activity are difficult to perform and interpret. To address this, we compared NF-κB activity in cultures of primary neocortical neurons, mixed brain cells, and liver cells, employing Western blot of NF-κB subunits, electrophoretic mobility shift assay (EMSA) of nuclear κB DNA binding, reporter assay of κB DNA binding, immunofluorescence of the NF-κB subunit protein p65, quantitative real-time polymerase chain reaction (PCR) of NF-κB-regulated gene expression, and enzyme-linked immunosorbent assay (ELISA) of produced proteins. Assay of p65 showed its constitutive presence in cytoplasm and nucleus of neurons at levels significantly lower than in mixed brain or liver cells. EMSA and reporter assays showed that constitutive NF-κB activity was nearly absent in neurons. Induced activity was minimal—many fold lower than in other cell types, as measured by phosphorylation and degradation of the inhibitor IκBα, nuclear accumulation of p65, binding to κB DNA consensus sites, NF-κB reporting, or induction of NF-κB-responsive genes. The most efficacious activating stimuli for neurons were the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-beta (IL-β). Neuronal NF-κB was not responsive to glutamate in most assays, and it was also unresponsive to hydrogen peroxide, lipopolysaccharide, norepinephrine, ATP, phorbol ester, and nerve growth factor. The chemokine gene transcripts CCL2, CXCL1, and CXCL10 were strongly induced via NF-κB activation by TNFα in neurons, but many candidate responsive genes were not, including the neuroprotective genes SOD2 and Bcl-xL. Importantly, the level of induced neuronal NF-κB activity in response to TNFα or any other stimulus was lower than the level of constitutive activity in non-neuronal cells, calling into question the functional significance of neuronal NF-κB activity.

A Deleterious Mutation in DNAJC6 Encoding the Neuronal-Specific Clathrin-Uncoating Co-Chaperone Auxilin, Is Associated with Juvenile Parkinsonism

Parkinson disease is caused by neuronal loss in the substantia nigra which manifests by abnormality of movement, muscle tone, and postural stability. Several genes have been implicated in the pathogenesis of Parkinson disease, but the underlying molecular basis is still unknown for ∼70% of the patients. Using homozygosity mapping and whole exome sequencing we identified a deleterious mutation in DNAJC6 in two patients with juvenile Parkinsonism. The mutation was associated with abnormal transcripts and marked reduced DNAJC6 mRNA level. DNAJC6 encodes the HSP40 Auxilin, a protein which is selectively expressed in neurons and confers specificity to the ATPase activity of its partner Hcs70 in clathrin uncoating. In Auxilin null mice it was previously shown that the abnormally increased retention of assembled clathrin on vesicles and in empty cages leads to impaired synaptic vesicle recycling and perturbed clathrin mediated endocytosis. Endocytosis function, studied by transferring uptake, was normal in fibroblasts from our patients, likely because of the presence of another J-domain containing partner which co-chaperones Hsc70-mediated uncoating activity in non-neuronal cells. The present report underscores the importance of the endocytic/lysosomal pathway in the pathogenesis of Parkinson disease and other forms of Parkinsonism.

An investigation of herpes simplex virus promoter activity compatible with latency establishment reveals VP16-independent activation of immediate-early promoters in sensory neurones.

Herpes simplex virus (HSV) type-1 establishes lifelong latency in sensory neurones and it is widely assumed that latency is the consequence of a failure to initiate virus immediate-early (IE) gene expression. However, using a Cre reporter mouse system in conjunction with Cre-expressing HSV-1 recombinants we have previously shown that activation of the IE ICP0 promoter can precede latency establishment in at least 30 % of latently infected cells. During productive infection of non-neuronal cells, IE promoter activation is largely dependent on the transactivator VP16 a late structural component of the virion. Of significance, VP16 has recently been shown to exhibit altered regulation in neurones; where its de novo synthesis is necessary for IE gene expression during both lytic infection and reactivation from latency. In the current study, we utilized the Cre reporter mouse model system to characterize the full extent of viral promoter activity compatible with cell survival and latency establishment. In contrast to the high frequency activation of representative IE promoters prior to latency establishment, cell marking using a virus recombinant expressing Cre under VP16 promoter control was very inefficient. Furthermore, infection of neuronal cultures with VP16 mutants reveals a strong VP16 requirement for IE promoter activity in non-neuronal cells, but not sensory neurones. We conclude that only IE promoter activation can efficiently precede latency establishment and that this activation is likely to occur through a VP16-independent mechanism.

Zhangfei, a novel regulator of the human nerve growth factor receptor, trkA

The replication of herpes simplex virus (HSV) in epithelial cells, and during reactivation from latency in sensory neurons, depends on a ubiquitous cellular protein called host cell factor (HCF). The HSV transactivator, VP16, which initiates the viral replicative cycle, binds HCF as do some other cellular proteins. Of these, the neuronal transcription factor Zhangfei suppresses the ability of VP16 to initiate the replicative cycle. It also suppresses Luman, another cellular transcription factor that binds HCF. Interactions of nerve growth factor (NGF) and its receptor tropomyosin-related kinase (trkA) appear to be critical for maintaining HSV latency. Because the neuronal transcription factor Brn3a, which regulates trkA expression, has a motif for binding HCF, we investigated if Zhangfei had an effect on its activity. We found that Brn3a required HCF for activating the trkA promoter and Zhangfei suppressed its activity in non-neuronal cells. However, in neuron-like NGF-differentiated PC12 cells, both Brn3a and Zhangfei activated the trkA promoter and induced the expression of endogenous trkA. In addition, capsaicin, a stressor, which activates HSV in in vitro models of latency, decreased levels of Zhangfei and trkA transcripts in NGF-differentiated PC12 cells.

Neuronal cell type-specific promoter of the α CaM kinase II gene is activated by Zic2, a Zic family zinc finger protein

To understand the neuronal cell type-specific expression of Ca 2+/calmodulin-dependent protein kinase II (CaM kinase II), we investigated binding proteins that specifically activated the promoter of the α isoform of CaM kinase II (α CaM kinase II). Proteins that bind the promoter sequence were found in rat brain nuclear extract by electrophoretic mobility shift assay. Then, we screened for binding proteins in a mouse brain cDNA library using the yeast one-hybrid system. Zic2, a Zic family zinc finger transcription factor, was identified as one of the binding proteins. To investigate the effect of Zic2 on the promoter activity, Zic2 cDNA was expressed with a luciferase reporter gene containing a neuronal cell type-specific promoter of α CaM kinase II in neuronal and non-neuronal cells. The promoter activity of α CaM kinase II was enhanced 1.3–5-fold in cultured neuronal cells by Zic2. The activation was varied among neuronal cell types. Zic2 also increased the promoter activity in non-neuronal cells, although the relative luciferase activites in non-neuronal cells were lower than those in neuronal cell lines. These results indicated that Zic2 was one of the proteins binding to, and regulating the activity of, the promoter of α CaM kinase II.

A neuroblastoma-selective suicide gene therapy approach using the tyrosine hydroxylase promoter.

In this study, selective expression of therapeutic transgenes was evaluated in neuroblastoma cells. Promoter fragments of the genes for neuron-specific enolase (NSEp), tyrosine hydroxylase (THp), and dopamine-beta-hydroxylase (DBHp) were studied in neuroblastoma and nonneuronal cell lines by transient transfection experiments using fluorescence-activated cell sorting (FACS) analysis of enhanced green fluorescent protein (egfp) and luciferase (luc+) assay. Both reporter gene assays revealed a neuroblastoma-selective expression mediated by NSEp and THp, whereas DBHp was active only in a murine neuroblastoma cell line. Reporter gene expression by NSEp in neuroblastoma cells was markedly higher than expression by THp, but NSEp also showed considerable background activity in nonneuronal cells. THp-driven expression of egfp was 35-fold higher in human neuroblastoma MHH-NB11 compared with nonneuronal HeLa cells. Thus, THp was chosen for a neuroblastoma-selective suicide gene therapy approach using the herpes simplex virus type 1 thymidine kinase (HSV-tk)/ganciclovir (GCV) system. A retrovirus vector that contained an expression cassette of a HSV-tk/egfp fusion gene and THp in antisense orientation was generated. Stably transduced human neuroblastoma cells and nonneuronal cell lines were generated, and HSV-tk/egfp expression was measured by FACS and GCV cytotoxicity assay. There was a 2.2-fold difference in green fluorescence and a 1.4-fold difference in cell killing between the human neuroblastoma MHH-NB11 and HeLa cells after HSV-tk/egfp gene transfer. The overall difference in THp-HSV-tk/egfp-mediated cell killing between neuroblastoma and nonneuronal tumor cell lines was statistically significant (P = 0.001). In conclusion, the present study demonstrated the feasibility of a neuroblastoma-selective gene therapy approach using the THp/HSV-tk/egfp expression cassette.

Molecular mechanisms regulating cell type specific expression of BMP/RA Inducible Neural-specific Protein-1 that suppresses cell cycle progression: roles of NRSF/REST and DNA methylation

We have recently identified a novel protein family, BMP/ RA- Inducible Neural-specific Protein (BRINP) including BRINP1, 2, 3. Among BRINP family genes, BRINP1 is most highly and widely expressed in various regions of the mammalian nervous system, although its expression is also found in some non-neural tissues and cell types at low levels. We have previously suggested that BRINPs are involved in the suppression of cell-cycle progression in post-mitotic neuronal cells. In the present study, we investigated the transcriptional mechanisms regulating the cell type-specific expression of BRINP1. First, bisulfite analysis of the methylation status revealed hypermethylation of the CpG island surrounding BRINP1 exon 1 in a non-neural cell line, NIH 3T3, which expresses low but detectable levels of BRINP1, while methylation levels of the BRINP1 CpG island in either non-neural or neural tissues are very low. Treatment of NIH 3T3 cells with a demethylating agent, 5-azacytidine, upregulated the expression of BRINP1 remarkably. Then, we analyzed the promoter activity of 7 kb region surrounding BRINP1 exon 1 in neuronal and non-neuronal cells. Consequently, we found a basic promoter region and a non-neural-specific silencing region which contains neuron-restrictive silencing element/repressor element 1 (NRSE/RE-1) like element (BRINP1-NRSE). Mutation of BRINP1-NRSE recovered the BRINP1 promoter activity in non-neuronal cells. Furthermore, proteins in nuclear extract from non-neural cells bound to the BRINP1-NRSE. These results strongly suggest that BRINP1-NRSE determines neural-specific expression of BRINP1, while hypermethylation of the BRINP1-CpG island suppresses BRINP1 expression in NIH 3T3 cells.

Mutational analysis of N-Bak reveals different structural requirements for antiapoptotic activity in neurons and proapoptotic activity in nonneuronal cells

N-Bak, a neuron-specific BH3-only splice variant of Bak, is proapoptotic when overexpressed in nonneuronal cells, but antiapoptotic in NGF-deprived sympathetic neurons. We generated mutants of N-Bak and compared their activities in COS-7 or Neuro2A cells to those in NGF-deprived sympathetic neurons. A C-terminal deletion shortly after the BH3 domain of N-Bak compromised its neuroprotective activity but had little effect on its cytotoxic activity in nonneuronal cells. Amino acid changes in the BH3 domain of N-Bak differently affected its function in nonneuronal cells and in neurons. The same changes in the BH3 domain of longer Bak isoform affected its function similarly in nonneuronal cells and neurons. C-terminally truncated Bax, a structural analogue of N-Bak, was also neuroprotective, whereas Blk, a different BH3-only protein was apoptotic in neurons. Thus, neuron-specific antiapoptotic interactions require a “N-Bak-type” conformation, not just a BH3 domain, whereas the presence of a BH3 domain in the Bak protein is sufficient to kill nonneuronal cells.

Role for RFX Transcription Factors in Non-neuronal Cell-specific Inactivation of the Microtubule-associated Protein MAP1A Promoter

Microtubule-associated protein MAP1A is expressed abundantly in mature neurons and is necessary for maintenance of neuronal morphology and localization of some molecules in association with the microtubule-based cytoskeleton. Previous studies indicated that its complementary expression together with MAP1B during nervous system development is regulated at the transcriptional level and that the mouse Map1A gene is transcribed under the control of 5' and intronic promoters. In this study, we investigated the regulatory mechanisms that govern the neuronal cell-specific activation of the MAP1A 5' promoter. We found that two regulatory factor for X box (RFX) binding sites in exon1 of both the mouse and human genes are important for effective transcriptional repression observed only in non-neuronal cells by reporter assays. Among RFX transcription factor family members, RFX1 and 3 mainly interact with repressive elements in vitro. Cotransfection studies indicated that RFX1, which is expressed ubiquitously, down-regulated the MAP1A 5' promoter activity in non-neuronal cells. Unexpectedly, RFX3, which is abundantly expressed in neuronal cells, down-regulated the transactivity as well, when it was expressed in non-neuronal cells. Both RFX1 and 3 did not down-regulate the transactivity in neuronal cells. These results suggest that RFX1 and 3 are pivotal factors in down-regulation of the MAP1A 5' promoter in non-neuronal cells. The cell type-specific down-regulation, however, does not depend simply on which RFX interacts with the elements, but seems to depend on underlying profound mechanisms.

Exposure to a solar eclipse causes neuronal death in the retina.

A solar eclipse was observed in Europe on 11 August 1999. Several individuals suffered from transient or persisting retinal damage, caused by gazing at the eclipse without adequate eye protection. Retinal damage is the most serious hazard of exposure to light. but the mechanisms by which this type of exposure produces retinal damage and its cellular correlates are not yet established. We used an animal model to monitor the mechanisms of retinal damage following excessive light exposure, and in particular to study whether observation of the eclipse induces death of retinal cells. In the geographic area where the experiment was conducted, a partial (90%) solar eclipse was observed. Experimental albino rats were exposed to these eclipse conditions, and control rats were exposed to normal sunlight. Another group of control animals was exposed to the same conditions, but was provided with protective light filters of the type recommended for human use. The DNA fragmentation in retinal sections of the various groups was analysed by terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labelling. This analysis revealed that exposure to both normal sunlight and to the eclipse resulted in neuronal apoptosis. Immunohistochemical techniques were used to evaluate possible glial-vascular alterations. Dying cells could first be detected 24 h after exposure, the largest number of which were found 6 days later in the photoreceptor layer. Control levels were attained 14 days after the exposure. Retinal ganglion cells underwent apoptosis in both groups (normal sunlight and eclipse exposure), whereas in the neuroglial cells there was an up-regulation of the intermediate filament content. The number of dying cells in both groups was greater in animals whose pupils had been dilated pharmacologically during exposure. On the other hand, the protective filters were effective in preserving the rat retinal cells from apoptosis. These results show, for the first time, that the cellular correlates of solar retinopathy are neuronal apoptosis accompanied by glio-vascular responses. Cellular apoptosis is an irreversible process, which could manifest itself as permanent visual impairment. The activation of non-neuronal cells, such as glial and endothelial cells, could be responsible for the more transient clinical symptoms.

Transcriptional control of the neuronal nicotinic acetylcholine receptor gene cluster by the β43′ enhancer, Sp1, SCIP and ETS transcription factors

Receptors assembled from the products of a neuronal β4α3α5 NAChR gene cluster depend on these genes being coordinately regulated in particular populations of neurons. Little is known, however, about the transcriptional mechanisms that are likely to underlie their co-expression in correct neuronal cell types. We have identified several regulatory elements and transcription factors that influence transcription of the α3 and β4 genes. The promoters of these genes appear to contain a common cis element that binds Sp1 transcription factors. They can be activated by the POU-domain factor SCIP and activation does not require SCIP binding sites. Between these two promoters is a cell type specific enhancer called β43′. This enhancer has little activity in non-neuronal cells and is preferentially active in particular populations of central neurons. The clustered genes are potential targets of ETS factors as the ETS domain factor, Pet-1 can activate β43′-dependent transcription. The neuron-selective properties of β43′ and its location suggest that it is a component of the cis regulatory information required to control expression of the β4 and α3 genes in specific populations of neurons.

Transcriptional Regulation of the GluR2 Gene: Neural-Specific Expression, Multiple Promoters, and Regulatory Elements

To understand how neurons control the expression of the AMPA receptor subunit GluR2, we cloned the 5' proximal region of the rat gene and investigated GluR2 promoter activity by transient transfection. RNase protection and primer extension of rat brain mRNA revealed multiple transcription initiation sites from -340 to -481 bases upstream of the GluR2 AUG codon. The relative use of 5' start sites was different in cortex and cerebellum, indicating complexity of GluR2 transcript expression among different sets of neurons. When GluR2 promoter activity was investigated by plasmid transfection into cultured cortical neurons, cortical glia, and C6 glioma cells, the promoter construct with the strongest activity, per transfected cell, was 29.4-fold (+/- 3.7) more active in neurons than in non-neural cells. Immunostaining of cortical cultures showed that >97% of the luciferase-positive cells also expressed the neuronal marker MAP-2. Evaluation of internal deletion and substitution mutations identified a functional repressor element I RE1-like silencer and functional Sp1 and nuclear respiratory factor-1 (NRF-1) elements within a GC-rich proximal GluR2 promoter region. The GluR2 silencer reduced promoter activity in glia and non-neuronal cell lines by two- to threefold, was without effect in cortical neurons, and could bind the RE1-silencing transcription factor (REST) because cotransfection of REST into neurons reduced GluR2 promoter activity in a silencer-dependent manner. Substitution of the GluR2 silencer by the homologous NaII RE1 silencer further reduced GluR2 promoter activity in non-neuronal cells by 30-47%. Maximal positive GluR2 promoter activity required both Sp1 and NRF-1 cis elements and an interelement nucleotide bridge sequence. These results indicate that GluR2 transcription initiates from multiple sites, is highly neuronal selective, and is regulated by three regulatory elements in the 5' proximal promoter region.

Cell Type-specific Regulation of Choline Acetyltransferase Gene Expression: ROLE OF THE NEURON-RESTRICTIVE SILENCER ELEMENT AND CHOLINERGIC-SPECIFIC ENHANCER SEQUENCES

This study demonstrates the presence of positive and negative regulatory elements within a 2336-base pair-long region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells. A 21-base pair-long neuron-restrictive silencer element (NRSE) was identified in the proximal part of this region. This element was recognized by the neuron-restrictive silencer factor (NRSF), previously shown to regulate expression of other neuron-specific genes. The ChAT NRSE was inactive in both cholinergic and non-cholinergic neuronal cells, but repressed expression from a heterologous promoter in non-neuronal cells. Specific deletion of this element allowed ChAT gene promoter activity in non-neuronal cells, and overexpression of NRSF repressed ChAT gene promoter activity in cholinergic cells. The distal part of the ChAT gene promoter showed cholinergic-specific enhancing activity, which stimulated promoter activity in cholinergic cells, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. Thus, at least two kinds of regulatory elements cooperate to direct ChAT gene expression to cholinergic neurons, namely a neuron-restrictive silencer element and a cholinergic-specific enhancer.

Activation of non-neuronal cells in the rat sciatic nerve in response to inflammation caused by implanted silicone tubes

The presence of macrophages and the induction of c-jun protein and proliferation of non-neuronal cells were studied following implantation of silicone tubes with different diameters (i.e. 0.8 or 1.6 mm) around the rat sciatic nerve. Three days after implantation, numerous EDI and ED2 positive macrophages could be observed around the nerve beneath the 1.6 mm tubes. Some EDI and ED2 positive macrophages were also present in the endoneurium. In contrast, there were numerous EDI and ED2 positive macrophages in the endoneurium beneath the tube and distally in nerves surrounded by the 0.8 mm tube. In these experiments, there was also a massive induction of c-jun protein and DNA synthesis in non-neuronal cells, as visualised by c-jun and BrdU antibodies respectively (e.g. a response similar to that observed after a crush lesion). Such activated cells, albeit few, were also present in the endoneurium beneath the tube of nerves with a 1.6 mm tube, but not distal to the tube in the endoneurium. At 7 days, the responses were somewhat amplified but essentially the same as at 3 days. The results showed that the large diameter implants, which do not cause axonal damage, as does the small diameter tube, but result in conditioning of the nerve [4], induced invasion of macrophages around the nerve and activation of some cells in the endoneurium beneath the tube. We suggest that cell activation is caused by factors released from macrophages and that endoneurial cell activation is important for the conditioning of the nerve by the silicone tube implant.