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.     

Ultrastructural immunocytochemical localization of neuron-specific enolase in parafollicular cells of the rat thyroid

Summary Using pre- and post-embedding procedures, neuron-specific enolase and calcitonin were localized in rat thyroid parafollicular cells by light and electron microscopy. Peroxidase-antiperoxidase (PAP), biotin-avidin (ABC) and protein A — colloidal gold techniques were used. In paraffin sections neuron-specific enolase was demonstrated in all calcitonin-storing parafollicular cells in rats aging 1 to 180 days. The post-embedding procedure failed to detect neuron-specific enolase in ultrathin sections, but the enzyme could be demonstrated using a preembedding procedure. Neuron-specific enolase was localized exclusively within the cytosol of parafollicular cells, while calcitonin was localized within secretory granules applying either post- or pre-embedding incubation techniques.Supported by Sonderforschungsbereich 232     

Immunohistochemical localization of neuron-specific enolase and calcitonin gene-related peptide in rat taste papillae.

Calcitonin gene-related peptide-like and neuron-specific enolase-like immunoreactivity (CGRP-IR and NSE-IR) were surveyed immunohistochemically in the fungi-form, foliate and circumvallate papillae in rats. A dense CGRP-IR network (subgemmal and extragemmal) in the taste papillae is linked to the presence of taste buds, even though CGRP-IR fibers are rarely present in the taste buds. Three typical fiber populations were detected with these two markers. (a) A population of coarse NSE-IR intragemmal fibers characterized by thick neural swellings, never expressing CGRP-immunoreactivity. (b) A population of thin varicose intragemmal NSE/CGRP-IR fibers. (c) A population of subgemmal and extragemmal NSE-/CGRP-IR fibers that partly penetrated the epithelium. The common distribution of CGRP-IR and NSE-IR fibers at the base of taste buds, their differential distribution and morphology within taste buds, added to their restricted nature (gustatory or somatosensory) suggest that a population of CGRP-IR fibers undergoes a target-induced inhibition of its CGRP phenotype while entering the taste buds. The combined use of NSE and CGRP allowed a better characterization of nerve fibers within and between all three types of taste papillae. NSE was also a very good marker for a subtype of taste bud cells in the foliate and in the circumvallate papillae, but no such cells could be observed in the fungiform papillae.     

Developmental changes of neuron-specific enolase mRNA in primary cultures of rat neurons

1. The level of mRNAs for neuron-specific enolase (NSE) and nonneuronal enolase (NNE) was studied in developing rat brain and in pure neuronal cultures of corresponding ages treated or not treated with triiodothyronine (T3). 2. In brain cortices both messages are already detectable at the earliest age (embryonal day 16; E16). During development the mRNA for NNE remains at a steady level, with a transient decline at postnatal day 5 (P5). 3. On the other hand, NSE mRNA follows a biphasic curve: the signal increases threefold from E-16 to P0 and threefold from P5 to P18, with a plateau between P0 and P5. 4. In neuronal cultures the NNE message is present at a constant level until day 10 and declines sharply thereafter, while in T3-treated cultures it reaches a minimum beforehand. 5. The NSE mRNA, on the other hand, increases continuously throughout the whole culture life span, and a slightly higher level is observed in T3-treated cells during the first ten days.     

Immunohistochemical localization of neuron-specific enolase and calcitonin gene-related peptide in pig taste papillae.

Immunoreactivity to neuron-specific enolase (NSE), a specific neuronal marker, and calcitonin gene-related peptide (CGRP) was localized in lingual taste papillae in the pigs. Sequential staining for NSE and CGRP by an elution technique allowed the identification of neuronal subpopulations. NSE-staining revealed a large neuronal network within the subepithelial layer of all taste papillae. NSE-positive fibers then penetrated the epithelium as isolated fibers, primarily in the foliate and circumvallate papillae, or as brush-shaped units formed by a multitude of fibers, especially in the fungiform papillae and in the apical epithelium of the circumvallate papilla. Taste buds of any type of taste papillae were found to express a dense subgemmal/intragemmal NSE-positive neuronal network. CGRP-positive nerve fibers were numerous in the subepithelial layer of all three types of taste papillae. In the foliate and circumvallate papillae, these fibers penetrated the epithelium to form extragemmal and intragemmal fibers, while in the fungiforms, they concentrated almost exclusively in the taste buds as intragemmal nerve fibers. Intragemmal NSE- and CGRP-positive fiber populations were not readily distinguishable by typical neural swellings as previously observed in the rat. The NSE-positive neuronal extragemmal brushes never expressed any CGRP-like immunoreactivity. Even more surprising, fungiform taste buds, whether richly innervated by or devoid of NSE-positive intragemmal fibers, always harboured numerous intragemmal CGRP-positive fibers. Consequently, NSE is not a general neuronal marker in porcine taste papillae. Our observations also suggest that subgemmal/intragemmal NSE-positive fibers are actively involved in synaptogenesis within taste buds. NSE-positive taste bud cells were found in all three types of taste papillae. CGRP-positive taste bud cells were never observed.     

Circulating neuron-specific enolase in medullary thyroid cancer

The utility of determining circulating neuron-specific enolase (NSE) in medullary thyroid carcinoma was assessed in 25 patients followed up for a mean period of 45.6 months. In 5 patients tested before any treatment serum NSE concentrations were in the normal range. After total thyroidectomy abnormally high serum NSE concentrations (more than 9.8 ng/ml) were found in 1/3 patients with normal calcitonin (CT) in remission, in 2/10 with elevated CT levels but no evidence of disease and in 9/12 with elevated CT levels and documented metastases. The mean (+/- SD) NSE value in this last group was 12.0 +/- 12.6 ng/ml, significantly higher than in the other groups (p less than 0.005). The time course of serum NSE in patients with long follow-up seems to indicate that serum NSE rises when a large tumor mass is present and usually parallels the pattern of circulating CT. Effective treatment of the metastases is usually followed by reduction of serum NSE. Thus, serum NSE can serve as an additional humoral marker for medullary thyroid carcinoma, its elevation being associated with important metastatic involvement and with a poor prognosis of the tumor.     

Immunocytochemical study on the localization of neuron-specific enolase and S-100 protein in the carotid body of rats

Light- and electron-immunocytochemical investigation with the peroxidase-antiperoxidase (PAP) procedure revealed neuron-specific enolase and S-100 protein-like immunoreactivities specifically localized in the chief cells and the sustentacular cells of the rat carotid body, respectively. This finding suggests a neuron-like nature of the chief cells and a glia-like nature of the sustentacular cells on both embryological and functional bases.     

Changes in levels of translatable mRNA for neuron-specific enolase and non-neuronal enolase during development of rat brain and liver

Neuron-specific enolase (NSE), and non-neuronal enolase (NNE) which exists in many tissues including liver but is localized in glial cells within the nervous system, were synthesized in the rabbit reticulocyte cell-free translation system programmed with brain mRNAs. The in vitro synthesized NSE and NNE were indistinguishable from the two enzymes purified from rat brains. NSE mRNA activity was found only in brain RNAs, while NNE mRNA activity existed in brain RNAs as well as liver RNAs. In developing brains, the level of translatable NSE mRNA was low at the embryonic stage and at birth, increased rapidly from about 10 days postnatal, and reached the adult level, while that of NNE mRNA was high at the embryonic stage and at birth, followed by a slight decrease then a gradual rise to adult levels. These changes correlated with the developmentally regulated appearance and accumulation pattern of each of the two enzymes. These results suggest that the levels of NSE and NNE are controlled primarily by the level of each of the two translatable mRNAs. In developing livers, only the NNE mRNA activity was detected and its level generally paralleled the changes in the level of NNE.     

Complete structure of the human gene encoding neuron-specific enolase

At least three genes encode the different isoforms of the glycolytic enzyme enolase. We have isolated the gene for the human gamma- or neuron-specific enolase and determined the nucleotide sequence from upstream to the 5' end to beyond the polyadenylation site. The gene contains 12 exons distributed over 9213 nucleotides. Introns occur at positions identical to those reported for the homologous rat gene, as well as for the human alpha- or nonneuronal enolase gene, supporting the existence of a single ancestor for the members of this gene family. Primer extension analysis indicates that the gene has multiple start sites. The putative promoter region lacks canonical TATA and CAAT boxes, is very G + C-rich, and contains several potential regulatory sequences. Furthermore, an inverted Alu sequence is present approximately 572 nucleotides upstream of the major start site. A comparison of the 5'-flanking region of the human gamma-enolase gene with the same region of the rat gene revealed a high degree of sequence conservation.     

Serum neuron-specific enolase in various pathological conditions.

Quantitative determination of neuron-specific enolase in the serum was performed by RIA method in 18 neurological patients and in 22 patients with pulmonary diseases. The data confirmed that the specificity of this marker is not absolute for the detection both of the nature and of the seat of origin of the disease. Further problems are posed in patients which simultaneously suffer from endocrine, nervous and pulmonary abnormality.     

Large scale preparation and crystallization of neuron-specific enolase

A simple method has been developed for the large scale purification of neuron-specific enolase [EC 4.2.1.11]. The method consists of ammonium sulfate fractionation of brain extract, and two subsequent column chromatography steps on DEAE Sephadex A-50. The chromatography was performed on a short (25 cm height) and thick (8.5 cm inside diameter) column unit that was specially devised for the large scale preparation. The purified enolase was crystallized in 0.05 M imidazole-HCl buffer containing 1.6 M ammonium sulfate (pH 6.39), with a yield of 0.9 g/kg of bovine brain tissue.     

Chromogranin-A as a serum marker for neuroendocrine tumors: comparison with neuron-specific enolase and correlation with immunohistochemical findings

BACKGROUND: Chromogranin-A (Cg-A) is a 439-amino-acid protein contained in secretory granules of neuroendocrine cells, in addition to specific hormone peptides or neuropeptides. Since Cg-A is co-released with peptide hormones its serum concentration can be used as a marker of neuroendocrine tumors. AIM: Evaluation of the analytical performance of a new IRMA method for Cg-A assay and of the clinical value of serum Cg-A and neuron-specific enolase (NSE) in neuroendocrine tumors. In addition, we compared the diagnostic usefulness of both Cg-A and NSE serum levels and their relationship to tissue expression. PATIENTS AND METHODS: Initially we evaluated the analytical performance (intra- and interassay imprecision, dilution test and detection limit) of the Cg-A RIACT method (CIS Bio-International, Gif-sur-Yvette, France). We selected 50 patients affected by various histologically confirmed neuroendocrine tumors (NETs): 111In-pentetreotide scan and helical computed tomography were employed to assess tumor extent. Cg-A and NSE were measured before surgery in serum samples of patients and 50 age-matched controls by IRMA methods. After surgery immunohistochemical stains for Cg-A and NSE were performed on surgical specimens of tumor tissue. RESULTS: Cg-A levels were significantly higher (p < 0.0001) in patients with NETs than in healthy controls and we found a positive correlation between serum and tissue expression (p < 0.05). Serum levels of Cg-A were also related to tumor extent (p < 0.05) but in some cases we observed significant elevation of serum Cg-A in small, intensely immunoreactive NETs. ROC curve analysis showed better accuracy for serum Cg-A compared to NSE in the diagnosis of NETs, while no significant relationship was found between serum expression and immunostaining for NSE. DISCUSSION: Our results confirmed the biological and clinical significance of circulating Cg-A as an expression of granular content in neuroendocrine tissues and supported the complementary usefulness of serum Cg-A in the diagnosis and evaluation of NETs together with imaging modalities.     

Immunohistochemical localization of S-100 protein,glial fibrillary acidic protein,and neuron-specific enolase in the pars distalis of quail,rat, and human hypophyses

Summary In the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.     

Immunohistochemical localization of S-100 protein, glial fibrillary acidic protein, and neuron-specific enolase in the pars distalis of quail, rat, and human hypophyses

In the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.     

Cytosolic levels of neuron-specific enolase in squamous cell carcinomas of the lung

To study the behavior and possible correlations of neuron-specific enolase (NSE) with other clinicobiological parameters, we measured the cytosolic levels of this marker by means of an immunoradiometric assay (IRMA) in 95 squamous cell lung carcinoma samples. We also analyzed the levels of pS2, tissue-type plasminogen activator (t-PA), hyaluronic acid (HA), free beta subunit of human chorionic gonadotropin (beta-HCG), CYFRA 21.1 and CA 125 in cytosol. On the cell surface we analyzed the concentrations of epidermal growth factor receptor (EGFR), HA, erbB-2 oncoprotein, CD44s, CD44v5 and CD44v6. Other parameters considered were clinical stage, lymph node involvement, histological grade (HG), ploidy and the cellular S-phase fraction measured by flow cytometry on nuclei obtained from fresh tissues. In the 95 squamous cell carcinomas the cytosolic levels of NSE varied from 4.5 to 2235 ng/mg protein (median: 267) and were significantly higher (p < 0.001) than those observed in 38 samples of normal pulmonary tissue obtained from the same patients (range: 56-657; median: 141.5). When classifying tumors according to the different parameters analyzed, we observed that the levels of NSE were higher in aneuploid than in diploid cases (p = 0.046) and in those that were HG3 than in those that were HG2 (p < 0.001). Tumors with high NSE levels (> 422 ng/mg protein; 75th percentile) were more likely to have high S-phase values (p = 0.012) and were more frequently aneuploid (p = 0.038) and HG3 (p < 0.001) than those with low levels of NSE (< 180 ng/mg protein; 25th percentile). These results lead us to the following conclusions: 1) the cytosolic concentrations of NSE are significantly higher in squamous cell carcinomas than in healthy pulmonary tissue, and 2) the cytosolic concentrations of NSE are not correlated with clinical stage or nodal involvement. However, in our study higher levels of the enzyme were statistically correlated with aneuploidy, histological grade 3 and S-phase. This may explain its association with poorer outcome and progression, but also the more favorable response of tumors with elevated NSE to chemotherapy, as suggested by other groups.     

Electron microscopic immunohistochemical localization of alpha-msh in the rat brain.

Using an immunohistochemical technique at the electron microscopic level, it has been shown that alpha-MSH is localized within the small vesicles of a few cell bodies found in the arcuate nucleus and numerous nerve fibers widely distributed throughout the brain. These findings suggest that alpha-MSH could possibly be considered as a neurotransmitter.     

Isolation of murine neuron-specific and non-neuronal enolase cDNA clones

cDNA clones corresponding to subunits of neuron-specific (gamma gamma and alpha gamma) and non-neuronal (alpha alpha) enolase isozymes were characterized from two mouse brain cDNA libraries. Our hybridization data revealed a partial homology of the coding sequences of mouse alpha, mouse gamma and rat gamma mRNAs. The noncoding sequences, however, appear to be specific for each mouse mRNA. Although coding for two polypeptides of the same molecular weight, the mRNA for the gamma subunit (2600 bases) is larger than that for the alpha subunit (1900 bases). The noncoding sequences for neuron-specific gamma mRNA (about 1300 bases) are therefore longer than those of the non-nervous tissue specific alpha mRNA (about 600 bases).