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PubMed1994

Axotomy induces intranuclear immunolocalization of neuron-specific enolase in facial and hypoglossal neurons of the rat.

Neuron-specific enolase as an enzyme of the glycolytic pathway is localized in the cytoplasm of nerve cells, but not in the cell nucleus. We have applied immunocytochemistry with 1:64,000 polyclonal anti-rat neuron-specific enolase to the brainstem of male and female adult Wistar rats following: (a) transection of the facial nerve with immediate microsurgical nerve suture (facial-facial anastomosis), (b) transection of the hypoglossal nerve with immediate suture (hypoglossal-hypoglossal anastomosis) and (c) transection of the facial and hypoglossal nerve with immediate suture of the proximal hypoglossal to the distal facial nerve stump (hypoglossal-facial anastomosis). Studying the intracellular immunolocalization of neuron-specific enolase in neurons of the facial and hypoglossal nucleus we detected that (1) in normal rats about 20% of all facial and hypoglossal neurons display not only cytoplasmic, but also intranuclear neuron-specific enolase-like immunoreactivity and (2) following any axotomy of the facial or hypoglossal peripheral nerve, the perikarya of all injured motoneurons react by an outstanding increase of neuron-specific enolase-like immunoreactivity in the karyoplasm. Similar findings were obtained in experiments on non-fixed cultured Neuro-2a cells that had been lesioned with hydrogen peroxide. Counting the absolute numbers of normal and reactive neurons at 1-365 days post axotomy revealed that the increase of neuron-specific enolase in neuronal cell nuclei is temporary and reversible. It is first detected at 2 days post axotomy, reaches its maximum at 10-18 days post axotomy and is no longer evident 56 days following surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Anastomosis, Surgical↗
PubMed1996

High neuron specific enolase levels in bronchoalveolar lavage fluid of patients with lung carcinoma: diagnostic value, relation to serum neuron specific enolase, and staging.

BACKGROUND: High levels of neuron specific enolase (NSE) have recently been described in the bronchoalveolar lavage (BAL) fluid of patients with lung carcinoma. Although its value in serum has been extensively studied, its diagnostic value in BAL fluid in terms of sensitivity, specificity, and predictive value have not been evaluated. In addition, its value in staging and relation to serum NSE are yet unknown. METHODS: NSE levels were determined on the same day in the BAL fluid and the sera of two groups of patients: those with newly diagnosed lung carcinoma and those with smoking related chronic obstructive pulmonary disease (COPD). Clinical TNM staging was also performed. Levels of NSE in BAL fluid were expressed as nanograms per 100 international units of lactate dehydrogenase. BAL fluid NSE levels of the two groups were compared with staging and serum NSE. RESULTS: A highly significant difference exists in BAL NSE in the two groups. For diagnostic purposes, the simultaneous measurements of serum NSE increases its sensitivity, but specificity remains unchanged. No correlation exists between BAL NSE and serum NSE, tumor size, nodal status, or the presence of metastases. BAL NSE is a better predictor of malignancy than serum NSE. CONCLUSION: BAL fluid measurements of NSE may have diagnostic value, specially if it is simultaneously measured in the serum. However, our study does not show any value for this technique in staging of lung carcinoma. Also it has no correlation with serum NSE. Studies will have to be performed to determine if BAL NSE can predict chemotherapeutic sensitivity.

Aged↗
PubMed1982

Localization of monoamine oxidases A and B in primate brains relative to neuron-specific and non-neuronal enolases.

Using serotonin and phenylethylamine deamination as measures of MAO A and MAO B activity respectively, positive correlations were observed between the activities of MAO A and MAO B in different areas of rhesus monkey and human brains. When the activities of MAO A and MAO B were compared with those of neuron-specific enolase and nonneuronal enolase (isozymes which are markers for neurons and glia), a slight but non-significant correlation was observed, suggesting that a simple distribution of MAO A in neurons and MAO B in glia is unlikely. This conclusion is supported by studies using synaptosomes, but contrasts with that from investigations of MAO from peripheral tissues, where experiments indicate that MAO A is predominantly localized in neurones.

Animals↗
PubMed1993

Characterisation of an epitope specific to the neuron-specific isoform of human enolase recognised by a monoclonal antibody raised against a synthetic peptide corresponding to the C-terminus of beta/A4-protein.

Antibodies to synthetic peptides corresponding to different regions of beta/A4-protein recognize deposits of amyloid in the brains of patients with Alzheimer's disease. Down's syndrome cases and in the normal ageing brain. We have prepared a monoclonal antibody, mAb 22.212, raised against a synthetic C-terminal peptide of beta/A4 protein (residues 28-40) which labelled senile plaques in Alzheimer's disease after proteolytic treatment of tissue sections. In addition to recognising synthetic beta/A4-peptides that include the C-terminal residues 28-42, the mAb 22.212 was found to cross-react with a soluble, 47 kDa protein found in brain homogenates. This protein was shown, by amino acid sequence analysis and immunoassay, to be neuron-specific enolase (NSE). The mAb 22.212 did not recognize the non-neuronal enolase (NNE) or muscle-specific enolase (MSE) isoforms and its epitope was mapped to a short stretch of amino-acids unique to NSE, near the C-terminus. The cross-reactive NSE epitope is sited between residues 402-423 in NSE and shows no common sequence with beta/A4, perhaps suggesting that it is a conformational epitope. The significance and applications of these findings are discussed.

Alzheimer Disease↗
PubMed1998

Serum neuron specific enolase: a marker for neuronal dysfunction in children with continuous EEG epileptiform activity.

Non-convulsive status epilepticus (NCSE) is a common complication of the childhood epileptic encephalopathies. An essential feature for the diagnosis of non-convulsive status epilepticus is a continuous epileptiform activity on the electroencephalogram (EEG). Dementia is thought to be a possible long-term sequel of non-convulsive status epilepticus, the mechanism of which has remained elusive. Neuron specific enolase is a marker of neuronal damage. The serum concentration of neuron specific enolase (sNSE) has been measured in 17 children with continuous epileptiform activity on the EEG and in 16 children with epilepsy but without a continuous dysrhythmia. There was a significant difference in the concentration of sNSE between the two groups.

Adolescent↗
PubMed1985

Factors involved in expression of neuron-specific and non-neuronal enolase activity in developing chick brain and in primary cultures of chick neurons.

The effect of various factors affecting non-neuronal enolase (NNE) and neuron-specific enolase (NSE) was investigated in developing brain of two different chick strains, in primary cultures of pure neurons and of mixed cultures of neuronal and glial cells. NNE and NSE activities reached their maximum at an earlier stage of brain development in the fast growing Hybro strain than in the Leghorn strain. In pure neurons cultured during 6 days, NNE was stimulated by hydrocortisone in presence or in absence of serum. Dibutyryl cyclic AMP (diBcAMP) stimulated NNE only in serum-free medium. NSE activity was increased by glial cell-conditioned medium in presence of serum and by removal of serum from the medium. Hydrocortisone and diBcAMP had no effect on NSE. In mixed cultures of neurons and glial cells both enolase activities were raised in absence of serum. Hydrocortisone and diBcAMP had no effect. Steroid hormones, insulin and serum albumin also modify both enolase activities in pure neurons and in mixed cultures of neurons and glial cells. Our results suggest that NNE and NSE are regulated separately by various factors involved in nerve cell maturation.

Animals↗
PubMed1985

Neuron-specific enolase in mucosal endocrine cells and carcinoid tumours of the small intestine: a comparative study with neuron-specific enolase immunocytochemistry and silver stains.

Endocrine cells of human small intestinal mucosa, small intestinal carcinoids and carcinoid liver metastases were stained with an immunocytochemical technique using an antiserum against neuron-specific enolase (NSE), with the argyrophil technique of Grimelius and with the argentaffin technique of Masson. In the normal mucosa, scattered NSE-immunoreactive cells were seen mainly in the deeper parts of the crypts. These cells, as shown in the same sections, corresponded to the argentaffin and/or argyrophil cells indicating that they were of endocrine type. All intestinal carcinoids (16 cases) displayed NSE immunoreactivity. However, this reaction did not correlate on the cellular level with the silver techniques employed. Thus, many tumour cells were NSE immunoreactive but lacked an argentaffin or argyrophil reaction and vice versa. On the light microscopical level the silver techniques reveal the presence of neurohormonal granules in the tumour cells, while the NSE immunoreactivity appears to disclose neuroendocrine differentiation of the tumour cells irrespective of their hormone and granular content. Out of 13 carcinoid liver metastases, eight displayed strong NSE immunoreactivity, three were weakly stained and two were unreactive. Consecutive or the same tumour sections showed an argentaffin and argyrophil reaction in all carcinoid metastases. Since silver staining provides one type of information and NSE immunocytochemistry another, they provide in combination a good discriminator for neuroendocrine tumours.

Carcinoid Tumor↗
PubMed1982

The development of immunoreactivity for neuron-specific enolase of preoptic and septal neurons in dissociated cultures.

The immunocytochemical visualization of neuron-specific enolase, which is a marker protein for differentiated neurons, was applied to follow the differentiation of preoptic and septal neurons in dissociated cultures. From 4 to 24 days in vitro, the relative numbers of stained neurons were counted and the staining intensity of individual neurons determined by absorbency measurements using a television-based densitometer. Whereas few stained cells could be observed at 4 DIV, 80% of the neurons were neuron-specific enolase-positive at 13 days in vitro. This value remained constant up to 24 days in vitro. The density of the immunoreaction product increased dramatically from 13 to 17 days in vitro and was still higher at 24 days in vitro. The glial and ependymal cells of the carpet, as well as neuroblasts, remained unstained. Comparison with morphological observations and immunocytochemical demonstration of neuronal peptides made earlier shows that expression of neuron-specific enolase closely parallels neuronal differentiation. These observations indicate that cultures derived from preoptic and septal neurons represent a viable model system for the study of neuronal maturation in vitro.

Animals↗
PubMed1986

Multiple calcium-activated neutral proteinases (CANP) in mouse retinal ganglion cell neurons: specificities for endogenous neuronal substrates and comparison to purified brain CANP.

Calcium-activated neutral proteinases (CANPs) and their specificities for axonally transported proteins were studied within intact axons of mouse retinal ganglion cell (RGC) neurons in vitro. Two CANP activities with markedly different properties were identified. CANP B, at endogenous calcium levels, selectively cleaved the 145,000 Da (145 kDa) neurofilament protein subunit to yield 143 and 140 kDa neurofilament proteins that are also major constituents of the axonal cytoskeleton. This process represents a posttranslational modification of the neurofilament protein subunit rather than the initial step in its degradation (Nixon et al., 1982, 1983). A second calcium-activated neutral proteinase activity, CANP A, appeared only when calcium levels in the incubating medium were 100 microM or higher. CANP A degraded most proteins in RGC axons but acted considerably more rapidly on high-molecular-weight species. In particular, a 290-320 kDa protein in the Group IV (SCb) phase of axoplasmic transport was degraded 3 X faster than other major axonal proteins, including neurofilament proteins and fodrin. When maximally expressed, CANP A activity represented an enormous proteolytic potential in RGC axons--more than 50% of the total axonal content of proteins larger than 60 kDa could be hydrolyzed within 5 min. The calcium requirements, inhibitor profile, and substrate specificity of CANP A were similar to those of mCANP, the major CANP of mouse brain purified to homogeneity, suggesting that these enzymes may be the same or highly related proteins. The existence in a single neuron type of two CANP activities with markedly different substrate specificities and enzymatic properties emphasizes the possible functional diversity of calcium-activated neutral proteinases in neurons. These functions include the posttranslational modification, as well as degradation of neuronal proteins.

Animals↗
PubMed1993

Neuron-specific splicing of the Alzheimer amyloid precursor protein gene in a mini-gene system.

Several forms of Alzheimer amyloid precursor protein (APP) mRNA are generated by alternative splicing. Among them, the APP695 mRNA skipping the exon 7 and 8 is expressed specifically in neurons, suggesting that this alternative splicing is regulated in a neuron-specific manner. As the first step for investigating the mechanism of the neuron-specific splicing, a mini-gene system was developed, in which mini-APP genes consisting of the exon 6, 7, 8, 9 and their flanking regions were introduced into neuronal and nonneuronal cultured cell lines to see their expression profiles. In the system the exon 7 and 8 of the mini-gene were significantly skipped in the neuronal cell, and the deletion study indicated that cis-acting elements for skipping the exons existed in the corresponding skipped-exon and its flanking regions. A small deletion upstream of the exon 8 suppressed the skipping of the exon 8 in the neuronal cell, suggesting that one of the regulatory sequence(s) for the exon skipping exists in a small region upstream of the skipped exon.

Alternative Splicing↗
PubMed1985

Distribution of neurofilament protein and neuron-specific enolase in peripheral neuronal tumours.

Peripheral neuronal tumours were studied by the peroxidase-antiperoxidase (PAP) method for the presence of the neurofilament protein (NFP) and neuron-specific enolase (NSE). All cases of ganglioneuromas and ganglioneuroblastomas were positive for NFP and NSE. Both markers were observed only in tumour cells showing differentiation towards ganglion cells. Of the 14 cases of neuroblastoma, 8 were positive for NFP and 12 were positive for NSE. NSE was detected in most neuroblastic tumour cells. However, NFP was found in neuroblasts with signs of differentiation, such as nuclear enlargement, but not in immature, small round cells. NFP was present in cell bodies as well as in cytoplasmic processes of partially differentiated neuroblasts. The majority of pseudorosettes showed no NFP stain. Thus, antibodies against both NFP and NSE are useful in the diagnosis of peripheral neuronal tumours. Moreover, the presence of NFP seemed to be related to the degree of tumour cell differentiation.

Brain Neoplasms↗
PubMed1991

Does steric interference between splice sites block the splicing of a short c-src neuron-specific exon in non-neuronal cells?

The neuron-specific splicing of the mouse c-src N1 exon was analyzed. Model src genes, transiently expressed in HeLa and LA-N-5 neuroblastoma cells, were assayed for the insertion of the 18-nucleotide neuron-specific N1 exon into their product mRNA. The normal clone fails to use this exon in HeLa cells but inserts the exon into 50% of the mature mRNA in LA-N-5 cells. When the exon and flanking intron sequences are placed between two adenovirus exons, the N1 exon is still only inserted in the neural cells. Thus, the neural specificity is a property of the exon itself and its immediate flanking sequences. Simply extending the length of the N1 exon to 109 nucleotides allows its efficient use in HeLa cells, implying that the exon is normally skipped because it is too short to allow spliceosomes to assemble at both ends simultaneously. This model predicts that exclusion of the exon should be sensitive to proteins or mutations that alter the relative strength of the flanking splice sites. Mutations that change these splice sites support this hypothesis.

Animals↗
PubMed1983

Expression of neuron-specific enolase in cultured neurons from the fetal rat.

A specific antiserum to neuron-specific enolase (NSE), an isoenzyme of the glycolytic enzyme enolase, has been used to immunocytochemically study the differentiation of dissociated embryonic brain cells grown in serum-supplemented or serum-free (defined) medium for 4-28 days. The number of positively stained neurons increased with time up to 21 days in culture, irrespective of the medium composition. By day 14, the majority of neurons contained immunoreactive NSE in their cell bodies and fiber profiles. These data indicate that cultured embryonic neurons undergo differentiation in their serum-supplemented or serum-free medium, and that dissociated brain cell cultures may provide a model system for investigating cellular and molecular aspects of neuronal differentiation.

Animals↗
PubMed2002

Adenoviral-mediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity.

Viral vectors are excellent tools for studying gene function in the brain, although a limitation has been the ability to effectively target transgene expression to specific neuronal populations. This generally cannot be overcome by the use of neuron-specific promoters, as most are too large to be used with current viral vectors and expression from these promoters is often relatively weak. We therefore developed a composite expression cassette, comprising 495 bp of the weak human SYN1 (synapsin-1) promoter and 800 bp of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Studies in hippocampal cultures, organotypic cultures, and in vivo showed that the 3' addition of the WPRE to the SYN1 element greatly increased enhanced green fluorescent protein expression levels with no loss of neuronal specificity. In vivo studies also showed that transgene expression was enhanced with no loss of neuronal specificity in dentate-gyrus neurons for at least 6 weeks following transfection. Therefore, unlike most powerful promoter systems, which mediate expression in neurons and glia, this SYN1-WPRE cassette can target powerful long-term transgene expression to central nervous system neurons when delivered at relatively low titers of adenovirus. Its use should therefore facilitate both gene therapy studies and investigations of neuronal gene function.

Adenoviridae↗
PubMed1980

Localization of neuron-specific enolase in mouse spinal neurons grown in tissue culture.

Neuron-specific enolase (NSE) is an isoenzyme of the glycolytic enzyme enolase (EC 4.2.1.11) which also has a muscle and liver isoenzyme. Previous work has shown NSE to be specifically localized to neurons and neuroendocrine cells, but the application of NSE as a marker for cell cultures has not been investigated. Primary culture of central nervous system tissue derived from mice have been used to study optimal fixation procedures. The results show that NSE can serve as a useful alternative to non-specific histochemical strains or strictly morphologic criteria for identifying nerve cells.

Animals↗
PubMed1992

Heterologous monoamine reuptake: lack of transmitter specificity of neuron-specific carriers.

The effect of systemic administration of desmethylimipramine (DMI), an inhibitor of the noradrenaline (NA) reuptake carrier, and of GBR 12909, an inhibitor of the dopamine (DA) reuptake carrier, on the in vivo extracellular concentrations of dopamine (DA) was studied by transcerebral dialysis in the prefrontal cortex and in the dorsal caudate of freely moving rats. In the NA-rich prefrontal cortex only DMI increased extracellular DA concentrations whereas in the dorsal caudate only GBR 12909 was effective. Haloperidol increased extracellular DA concentrations more effectively in the dorsal caudate than in the prefrontal cortex. Pretreatment with DMI, which failed to modify the effect of haloperidol in the dorsal caudate, potentiated its action in the prefrontal cortex. The reverse was obtained after GBR 12909+ haloperidol in the two areas. 6-hydroxydopamine lesioning of the dorsal NA bundle prevented the ability of DMI to increase DA concentrations. The results suggest that reuptake into NA terminals is an important mechanism by which DA is cleared from the extracellular space in a NA-rich area such as the prefrontal cortex. The elevated extracellular concentrations of DA resulting from blockade of such mechanism by tricyclic antidepressants may play a role in the therapeutic effects of these drugs.

Animals↗
PubMed1980

Developmental profile of neuron-specific (NSE) and non-neuronal (NNE) enolase.

Neurons and glia of mature brain can be distinguished by their isoenzyme content of the glycolytic enzyme enolase. Neurons contain neuron-specific enolase (NSE) and glial cells have non-neuronal enolase (NNE). Measurement of each isoenzyme by specific radioimmunoassay during the course of brain development in rat shows that NSE levels are very low in embryonic brain and increase at a time coincident with the morphological and functional maturation of neurons. NNE levels are high in embryonic brain and decrease when NSE first appears, followed by a gradual increase to adult levels. NSE levels rise at a slower rate in brain areas known to develop over a more protracted period (forebrain, cerebellum) compared to areas that develop more rapidly (brain stem). The data are consistent with a hypothesized switch from NNE to NSE during neuronal development. In E60 and E100 monkey brain tissue NSE/NNE ratios are higher in regions containing older neurons. This suggests that a similar switch from NNE to NSE also occurs during neuronal development in monkey.

Aging↗
PubMed1993

Herpesvirus-mediated gene delivery into the rat brain: specificity and efficiency of the neuron-specific enolase promoter.

1. Herpesvirus infection with genetically engineered vectors is a way to deliver foreign gene products to various cell populations in culture and in vivo. Selective neuronal gene expression can be achieved using the neuron-specific enolase (NSE) promoter regulating expression of a transgene placed in and delivered by a herpesvirus vector. 2. We sought to determine the anatomical specificity and efficiency of herpesvirus-mediated gene transfer into the rat brain following placement of virus particles carrying a transgene (lacZ) under control of the NSE promoter. The virus utilized was thymidine kinase (TK) deficient and therefore replication deficient in the brain. 3. Infusion of 10(6) plaque-forming units of virus into the striatum caused a limited number of striatal neurons to express the lacZ transgene mRNA and protein product 7 days postinfection. In addition, small numbers of neurons expressing the transgene mRNA and protein were found ipsilateral to the viral injection in the frontal cortex, substantia nigra pars compacta, and thalamus. Neurons at these anatomic loci project directly to the striatal injection site. No other cells within the brains of injected animals expressed the lacZ gene. 4. While this herpesvirus NSE vector was capable of introducing novel functional genetic information into postmitotic neurons within defined neuroanatomic constraints, the numbers of neurons expressing detectable levels of beta-galactosidase was minimal. The calculated efficiency of delivery and transgene expression at 7 days postinfection was 1 transgenic neuron per 10(4) virus particles infused. 5. We conclude that NSE probably is not an optimal promoter for use in gene delivery to CNS neurons in herpesvirus vectors and that the efficacy of gene delivery using other neuron-specific promoters placed at various sites in the herpes viral genome needs to be explored.

Animals↗