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Electron microscopic localization of neuron-specific enolase in rat and mouse brain.

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.

Animals↗

Sensory responsiveness of "broad-spike" neurons in the laterodorsal tegmental nucleus, locus coeruleus and dorsal raphe of awake rats: implications for cholinergic and monoaminergic neuron-specific responses.

Although cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei have been shown to have a pivotal role in neural mechanisms of paradoxical sleep, their function during wakefulness is less understood. To examine the latter, we have recorded from "broad-spike neurons", which were distinguished by their long spike duration, in the laterodorsal tegmental nucleus of undrugged, head-restrained rats, and examined their response properties to sensory stimuli such as light touch to the tail, air puff to the face, 2 kHz pure tone and flashes of light. Broad-spike neurons from the locus coeruleus and dorsal raphe nucleus were studied for comparison; these neurons have been demonstrated to be noradrenergic and serotonergic, respectively. The broad-spike neurons in the laterodorsal tegmental nucleus have also been suggested to be cholinergic. There were two kinds of responses: (1) a simple increase or decrease in firing, reflecting an elevated level of vigilance; and (2) a phasic response composed of a single spike or brief, high frequency burst, usually diminishing or disappearing upon repetition of the stimulus. When two or more types of stimuli were effective in a neuron, they evoked responses of the same quality. Most of the dorsal raphe neurons displayed only the simple increase of firing, whereas the locus coeruleus neurons gave a phasic response with rather weak attenuation upon repetition. Compared with these, the laterodorsal tegmental neurons were heterogeneous: about one-quarter showing only a simple change of firing (half increasing, half decreasing); and two-thirds displaying phasic responses. The latter response of many neurons attenuated strongly upon repetition. The laterodorsal tegmental neurons were classified into several groups according to their spontaneous firing behavior during sleep and wakefulness, but every neuron in a group did not show the same type of response. For example, some of the neurons which were most active during paradoxical sleep and essentially silent during wakefulness decreased or stopped firing upon sensory stimulation, while others in this group had strong phasic responses. These results suggest that putative cholinergic neurons in the laterodorsal tegmental nucleus have heterogenous properties not only with respect to their spontaneous activity during sleep and wakefulness but also with respect to their response to sensory stimulation. Some of these neurons may function to induce a global attentive state in response to a novel stimulus.

Acoustic Stimulation↗

Target neuron-specific process formation by embryonic mesencephalic dopamine neurons in vitro.

Mesencephalic dopamine neurons from the embryonic mouse brain were dissociated, aggregated in vitro in the presence of dissociated cells from appropriate or inappropriate target neuron areas, and visualized by the Falck-Hillarp histofluorescence technique after exposure to 1 microM exogenous dopamine. When aggregated with the surrounding rostral mesencephalic tegmentum cells only or with the addition of rostral tectum cells, the dopamine neurons formed a dense dendritic arborization, but no axons were observed. In the presence of dopamine-neuron target cells from the corpus striatum, a dense axonal plexus characteristic of that formed in this area in vivo was observed. In contrast, in aggregates formed with target cells from the frontal cortex, branching fluorescent axons bearing irregularly spaced and shaped varicosities were found coursing through the neuropil, as is characteristic of the dopaminergic innervation to the frontal cortex in vivo. Only proximal dendrites were observed in the presence of these axonal target cells. Dopamine neurons cultured with inappropriate target cells from the occipital cortex did not form either extensive axonal or dendritic processes. Thus, the presence, type, and distribution of dopamine neuronal processes are dependent on the presence of appropriate target cells. The formation of unique patterns of neuronal processes by dissociated neurons in vitro suggests that the information necessary for this differentiation is intrinsic to the dopamine neurons and their target cells. This system provides a useful model with which to study basic mechanisms underlying neuronal recognition.

Animals↗

Adult-specific neurons in the nervous system of the moth, Manduca sexta: selective chemical ablation using hydroxyurea.

The segmental ganglia of adults of the moth, Manduca sexta, are constructed both from remodeled larval neurons and from adult-specific cells. The latter are produced by identified stem cells (neuroblasts) during larval life and then differentiate to form functional neurons during metamorphosis. The mitotic activity of the larval neuroblasts could be irreversibly blocked by the DNA-synthesis inhibitor hydroxyurea (HU). Treatment on day 1 of the third larval stage resulted in 80-90% of the neuroblasts being blocked before they produced any progeny while leaving the functional larval neurons unaffected. Treated larvae finished growth, underwent metamorphosis, and produced an adult CNS that contained the normal set of remodeled larval neurons but lacked most of the new adult-specific cells. When HU treatment was delayed until the start of the fourth or fifth larval stage, the neuroblasts produced the early portions of their respective lineages before they were blocked. The immature neurons that were generated prior to treatment survived to contribute adult-specific neurons to the moth CNS, but the remainder of each lineage was missing. This technique therefore enables one to produce adult nervous systems containing the basic set of remodeled larval cells plus defined sets of adult-specific neurons.

Animals↗

Immunohistochemistry of neuron-specific and glia-specific proteins.

This paper reviews the immunohistochemical distribution of four brain-derived proteins, NSE, NFP, spot 35 and S-100 protein, in neuronal and paraneuronal tissues, concentrating on the results of our own research group. Neuron-specific enolase (NSE), a brain-specific isozyme of the glycolytic enzyme enolase, is characterized by its consistent occurrence in the cytoplasm of mature neurons. Immunoreactivity for NSE has been found in almost all paraneurons of both sensory and endocrine nature, suggesting that a unique system of intracellular energy metabolism may be shared by neurons and paraneurons. Neurofilament protein (NFP), a neuronal cytoskeletal protein, is immunohistochemically recognized in only a part of neurons, apparently due to the scarcity in neurofilaments in some neurons or to a decreased antigenicity for NFP in the cell. Similarly, only small populations of cells in restricted types of paraneurons, including gut endocrine cells and thyroid C cells, are immunoreactive for NFP. However, the potentiality for paraneurons to express NFP is given credence by the fact that its immunoreactivity is rather frequently found in the neoplasmas of paraneurons which normally do not show this immunoreactivity. Spot 35 protein which is a cerebellar Purkinje cell-specific protein, has displayed immunohistochemical localization in sensory and endocrine paraneurons as well as some neurons including a specific type of intramural nerve cells in the gut. The selective localization of this calcium-binding protein may be correlated with the secretory function and calcium-dependent excitable membrane property in those cells. S-100 protein, another brain-derived calcium-binding protein, is contained in sustentacular cells of paraneuronal organs as well as glial elements of nervous systems. Although it is now widely accepted that S-100 protein is not "glia-specific" but contained in a variety of non-glial cells, its beta subunit protein is still a useful marker for a series of glial and sustentacular cells.

Animals↗

Upregulation of the neuron-specific K+/Cl- cotransporter expression by transcription factor early growth response 4.

The expression of the neuron-specific K+/Cl- cotransporter (KCC2) is restricted to the CNS and is strongly upregulated during neuronal maturation, yielding a low intracellular chloride concentration that is required for fast synaptic inhibition in adult neurons. To elucidate the mechanisms of KCC2 gene regulation, we analyzed the KCC2 (alias Slc12a5) promoter and proximal intron-1 regions and revealed 10 candidate transcription factor binding sites that are highly conserved in mammalian KCC2 genes. Here we focus on one of these factors, early growth response 4 (Egr4), which shows a similar developmental upregulation in CNS neurons as KCC2. KCC2 luciferase reporter constructs containing the Egr4 site (Egr4(KCC2)) were strongly induced by Egr4 overexpression in neuro-2a neuroblastoma cells and in cultured neurons. Egr4-mediated induction was decreased significantly by point-mutating the Egr4(KCC2). Insertion of Egr4(KCC2) into the KCC2 basal promoter in the endogenous reverse, but not in the opposite, orientation reestablished Egr4-mediated induction. Electrophoretic mobility shift assay confirmed specific Egr4 binding to Egr4(KCC2). Interference RNA-mediated knock-down of Egr4 and a dominant-negative isoform of Egr4 significantly inhibited KCC2 reporter induction and endogenous KCC2 expression in cultured neurons. Together, the results indicate an important role for Egr4 in the developmental upregulation of KCC2 gene expression.

Animals↗

Auditory neuropathy in hyperbilirubinemia: is there a correlation between serum bilirubin, neuron-specific enolase levels and auditory neuropathy?

This study evaluated whether a correlation exists between increased serum bilirubin and neuron-specific enolase (NSE) assays (a biochemical index of neuronal damage) and auditory neuropathy. Nineteen term neonates without hemolysis whose serum bilirubin levels were above 20 mg/dl and 27 healthy term newborns with bilirubin levels <13 mg/dl were included in the study. Auditory brainstem responses (ABRs) and transient evoked otoacoustic emissions (TEOAEs) of patients with hyperbilirubinemia were obtained before discharge. This preliminary study did not show any correlation between the serum NSE and bilirubin values. However, infants who had auditory neuropathy had significantly higher NSE levels, and thus these patients, being in the high-risk group, need close follow-up.

Auditory Diseases, Central↗

Neurone-specific enolase persists in some hippocampal cells 24 hours after an NMDA injection.

Neurone-specific enolase immunoreactivity (NSE-IR) was examined in the hippocampal dentate gyrus 5 h, 24 h and 6 days following local injection of N-methyl-D-aspartate (NMDA), and was compared with Nissl substance and acid fuchsin staining. Five and 24 hours post-injection extracellular NSE-IR was increased, and the number of NSE-IR neurones in the granule cell layer was decreased. However, at 24 h post-injection only, 10-15% of the granule cells were strongly NSE-IR, in contrast to the Nissl/acid fuchsin staining which showed a homogeneous population of degenerating neurones. By 6 days there were no viable neurones in the lesion area stained by either method. These results show that neuronal degeneration after an excitotoxic insult occurs in a non-uniform way in an apparently homogeneous group of central neurones.

Animals↗

Neuron-specific expression of neuroglobin in mammals.

Neuroglobin, a vertebrate oxygen-binding protein, is expressed in many regions of the adult brain. We examined the cell type-specific expression of neuroglobin in neurons and astroglial cells in primary cultures of fetal hippocampal cells and sections of the adult mouse brain using neuroglobin-specific polyclonal antibodies and cell type-specific markers NeuN and GFAP to differentiate between neurons and glial cells. Neuroglobin is exclusively expressed in neurons, but not in astroglial cells. Accordingly, neuroglobin was detected in two neuroblastoma cell lines (N2a, SH-SY5Y) and the pheochromocytoma cell line PC-12, but not in glioblastoma cell lines (DKMG, GAMG) or other, non-neural cells (HeLa, Vero). Analysis of the neuroglobin genomic sequence from man and mouse identifies sequence motifs with similarity to the neuron-restrictive silencer element, possibly explaining a neuron-specific expression of neuroglobin.

Animals↗

Simultaneous expression of glial fibrillary acidic (GFA) protein and neuron-specific enolase (NSE) by the same reactive or neoplastic astrocytes.

In normal cells of the central nervous system (CNS), glial fibrillary acidic (GFA) protein is demonstrable by immunohistochemistry in fibrillated astrocytes, and neuron-specific enolase (NSE) in neurons and their processes. However, it has been shown that NSE may also be expressed in reactive astrocytes and in various neoplastic cells of non-neuronal origin, including those of astrocytomas and glioblastomas. In the present study, a double-labelling technique using immunoperoxidase (PAP) and immunofluorescence (FITC) was employed to determine whether GFA protein and NSE could be expressed simultaneously by the same cell. This was found to be the case in some, but not in all reactive astrocytes in the human brain. In glial tumours, many of the neoplastic cells in the glioblastomas and astrocytomas examined demonstrated either GFA protein or NSE, but usually not both. However, occasional neoplastic cells in those gliomas were found to show both proteins. Because of the relatively low sensitivity of the FITC technique, the percentage of cells expressing both proteins could not be determined, but it is clearly possible for a single reactive or neoplastic astrocyte to demonstrate both GFA protein and NSE.

Antigen-Antibody Reactions↗

Adenoviral-mediated neuron specific transduction of angiotensin II type 2 receptors.

The angiotensin II (Ang II) type 2 receptor (AT2R) is localized at specific nuclei within adult rat brain. However, a lack of specific approaches for manipulating the activity of neuronal AT2R has meant that the physiological actions of these sites in the brain remain to be established. Therefore, in this study, our aim was to develop a method by which AT2R can be specifically overexpressed in neurons and in rat brain, with the ultimate goal of a producing a system where discrete increases in AT2R levels in brain nuclei could reveal (and be linked to) physiological actions. Here, we have constructed an AT2R recombinant adenoviral vector, Ad5-SYN-AT2R-IRES-EGFP, which contains the AT2R gene and an IRES-linked EGFP reporter gene, both driven by the neuron-specific synapsin I (SYN) gene promoter. This vector efficiently transduces the AT2R into neuronal cells in culture and results in the expression of high levels of AT2R. These expressed receptors are functional in terms of inhibition of Erk mitogen activated protein kinases (Erk MAPK) and stimulation of neuronal K+ current. Furthermore, microinjection of this vector into adult rat brain elicits a long lasting ( approximately 1 month) expression of AT2R within neurons. In summary, we have developed a viral vector that can be used for the efficient transduction of AT2R into neurons both in vitro and in vivo, the use of which may help to define the physiological functions of brain AT2R in adult rats.

Adenoviridae↗

Characterization of BHC80 in BRAF-HDAC complex, involved in neuron-specific gene repression.

BRAF-HDAC complex (BHC) has been shown to contain six components, including BHC80, and to mediate REST-dependent transcriptional repression of neuron-specific genes in non-neuronal cells. In this study, we have examined the functional role(s) of BHC80 in mouse tissues and human cultured cells. Two isoforms of mouse BHC80 were predominantly present in the central nervous system and spermatogenic cells. Human cultured cells also contained two isoforms of BHC80. Immunohistochemical analysis showed the presence of mouse BHC80 in the nucleus of neuronal cells in the hippocampus and cerebellum. The C-terminal region of human BHC80 containing PHD zinc-finger domain was capable of binding directly to each of five other components of BHC, and of organizing BHC mediating transcriptional repression. Moreover, two isoforms of human BHC80 were distinguished from each other by reduced binding to HDAC1 and HDAC2, despite the presence of the PHD finger domain. These results suggest that BHC80 presumably serves as a scaffold protein in BHC in neuronal as well as non-neuronal cells. A possible role of BHC80 in spermatogenesis is also suggested.

Alternative Splicing↗

Multiple neuron-specific enhancers in the gene coding for the human neurofilament light chain.

To define DNA regions involved in the neuron-specific expression of the neurofilament light (NF-L) gene, we generated transgenic mice bearing different NF-L constructs. A 4.9-kilobase human NF-L fragment including -292 base pairs of 5'-flanking sequences contained sufficient elements for nervous system expression in transgenic mice. Deletion of introns 1 and 2 from this 4.9-kilobase DNA fragment resulted in reduced levels of transgene expression in the cortex, while deletion of intron 3 had little effect. Both introns 1 and 2 could act independently as enhancers to confer neuronal expression of the basal heat shock promoter (hsp68) fused to lacZ in transgenic mice. The hNF-L basal promoter (-292 base pairs) was found to contain elements for directing neuronal expression of either the lacZ reporter gene or an intronless hNF-L construct. Sequence comparison revealed that intron 1, intron 2, and the basal human NF-L promoter all contain an ETS-like motif, CAGGA, present in a variety of genes expressed in the nervous system.

Animals↗

Serum neuron-specific enolase in children with neuroblastoma. Relationship to stage and disease course.

Serum neuron-specific enolase (NSE) was measured in 61 children at diagnosis with all stages of neuroblastoma. The median serum values for Stages I, II, III, IV, and IV-S were 13, 23, 40, 214, and 40 ng/ml, respectively. Mean serum levels were different between groups I versus IV, (P = 0.0004) II versus IV (P = 0.0001) and IV-S versus IV (P = 0.004). The prognostic value of serum NSE for disease-free survival was determined in 54 patients at risk for relapse 2 or more years after diagnosis. The disease-free survival rate of all patients with levels of less than 100 ng/ml was 27/34 (79%), whereas it was 2/20 (10%) for those with higher levels. In 28 patients with lower stage disease and a good prognosis (Stages I, II, and IV-S) NSE levels were not predictive of relapse. Only 1 of these 28 patients had a raised level (greater than 100 ng/ml) and survived without relapse, whereas 4 patients who relapsed had serum NSE less than 100 ng/ml at diagnosis. In patients with Stages III and IV disease, a raised serum NSE level was associated with poor outcome: only 1/19 (5%) survived with NSE levels greater than 100 ng/ml, whereas survival was 5/8 (63%) with values below 100 ng/ml. Serial samples were analyzed on 17 patients; all 8 patients with initial NSE levels greater than 100 ng/ml achieved near normal levels during remission (median, 21 ng/ml). However, in only 4/10 patients studied at time of relapse, did the levels rise coincident with relapse. The sera of 47 patients with other forms of cancer and 19 siblings of cancer patients were at or near the normal limits (0-15 ng/ml), with three exceptions: acute lymphoblastic leukemia (286 ng/ml), hepatoblastoma (176 ng/ml), and primitive neuroectodermal tumor (105 ng/ml). Serum NSE is a useful marker for patients with advanced neuroblastoma in whom elevated levels were associated with a poor outcome; the raised NSE levels returned to near normal after therapy. In patients with Stage IV-S disease serum NSE levels were significantly lower than those in Stage IV despite their extensive tumor burden. Serum NSE estimation may confirm Stage IV-S status and suggest a more benign clinical course.

Carcinoma, Hepatocellular↗

Immunohistochemical positivity for neuron-specific enolase and Leu-7 in malignant mesotheliomas.

The discovery of immunostaining for neuron-specific enolase (NSE) and Leu-7 in a small cell mesothelioma prompted us to study some putative immunohistochemical markers of neuroendocrine differentiation in malignant mesotheliomas and to examine any diagnostically important immunohistochemical distinctions or similarities between malignant mesothelioma and other histologically similar lung tumours. Most mesotheliomas were positive for NSE (96 per cent) and Leu-7 (70 per cent) and positivity for these two markers was also found in small cell carcinomas (NSE 25 per cent, Leu-7 81 per cent) and adenocarcinomas (NSE 28 per cent, Leu-7 28 per cent) but carcinosarcomas were positive for only NSE (44 per cent). Chromogranin A positivity was found only in occasional small cell carcinomas (6 per cent) and adenocarcinomas (6 per cent). No tumour was positive for bombesin. The high incidence of NSE and Leu-7 positivity in mesotheliomas is an important original observation because it guards against the unjustified exclusion of mesothelioma from a differential diagnosis on the basis of positivity for these two markers.

Antigens, Differentiation↗

Neuron-specific enolase and malignant lymphomas (23 cases).

The immunoreactivity of polyclonal antiserum to neuron-specific enolase (NSE) has been investigated. Twenty-three cases of malignant lymphoma (ML) were studied and compared with previously published reports. In our study 11 out of 23 cases showed strong or weak NSE positivity; any type of ML could be positive or negative even among B or T cell ML. This study indicated that polyclonal NSE is not a specific marker; it might be an inconstant marker of ML with no apparent correlation between reactivity and morphology or phenotype.

Antibodies, Monoclonal↗