FUNCTIONAL PROPERTIES OF NEURONAL AND GLIAL ISOENZYMES OF BRAIN ENOLASE

Two of the major brain enolase (EC 4.2.1.11) isoenzymes exist as cell specific forms. The neuron specific enolase (NSE) is localized in neurons and the non-neuronal enolase (NNE) in glial cells. A third enolase containing one subunit from each of the above species is also present in brain and has been designated hybrid enolase. The stabilities of the brain enolases towards incubation with chloride and bromide salts is markedly different. NNE is rapidly inactivated upon incubation in 0.5 M-KCI or KBr while NSE is minimally effected and the hybrid has an intermediate stability. The inactivation is temperature dependent and reversible by salt removal. Magnesium exerts a stabilizing effect on each enzyme form. The mechanism of the reversible salt inactivation involves dissociation of the enolase subunits with reassociation occurring during reactivation. The brain enolases also display marked stability differences during incubation in 3 M-urea. with the neuronal form again being more stable. The urea inactivation was highly reversible for NNE but only marginally so for NSE. The neuronal enolase is also by far the most stable of the brain enolases at 50°C.     

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.     

A More Sensitive Radioimmunoassay for Neuron-Specific Enolase Suitable for Cerebrospinal Fluid Determinations

Abstract: Neuron-specific enolase (NSE) and non-neuronal enolase (NNE) have been shown to be highly specific neuronal and glial products respectively and are therefore useful as biochemical markers of the two major cell types in the vertebrate central nervous system. An iodinated radioimmunoassay (RIA) procedure for human NSE (NSE-H) with approximately 50-fold greater sensitivity than the previously available tritiated assay is described. This assay is capable of detecting 100 pg of NSE-H per assay. NSE levels in human cerebrospinal fluid (CSF) which were previously undetectable with the tritiated RIA are now easily measured and have been shown to be approximately 2 ng/ml of CSF. Furthermore, results obtained with the newly described assay procedure on more concentrated brain tissue extracts are comparable to the tritiated RIA. The iodinated NSE RIA is also shown to be capable of accurately detecting added amounts of NSE in human CSF, indicating the potential clinical usefulness of this assay in determining elevated levels of NSE in CSF.     

Brain Levels of Neuron-Specific and Nonneuronal Enolase in Huntington's Disease

Abstract— Levels of the cell-specific brain isoenzymes of enolase were determined in basal ganglia and cerebral cortical tissue of Huntington's disease and age- and sex-matched control brain. Neuron-specific enolase (NSE) levels are decreased an average of 45% in basal ganglia from patients with Huntington's disease whereas the glial-specific form of enolase, nonneuronal enolase (NNE), is not significantly altered. In contrast, levels of NSE in cerebral cortical tissue from Huntington's disease patients remains unchanged in comparison with controls whereas NNE levels are significantly increased. NNE and NSE levels appear to be specific biochemical indicators of glial and neuronal cell number and viability. Levels of these cell-specific isoenzymes may therefore prove useful in quantitating neuropathological changes in various neurological disorders.     

Blood Platelets Contain a Neuron-Specific Enolase Subunit

Abstract: Neuron-specific enolase (NSE) is a cell-specific isoenzyme of the glycolytic enzyme enolase that is present only in neurons and selected neuroendocrine cells. We now report the presence of this neuronal marker in blood platelets. The level of NSE found in human blood platelets is much lower than that found in brain tissue (0.045% of the total soluble protein for platelets versus 1.5% for cortical tissue), but is 20-30 times higher than NSE levels found in peripheral non-nervous tissues. Chromatographic analysis indicates that the majority of the NSE γ-subunit in platelets is present as the hybrid αγ isoenzyme. This, coupled with the high level of non-neuronal enolase (NNE) found in platelets, indicates that blood platelets contain both the α- and γ- subunit.     

Molecular cloning and the nucleotide sequence of cDNA to mRNA for non-neuronal enolase (alpha alpha enolase) of rat brain and liver.

The nucleotide sequence for alpha alpha enolase (non-neuronal enolase: NNE) of rat brain and liver was determined from recombinant cDNA clones. The sequence was composed of 1722 bp which included the 1299 bp of the complete coding region, the 108 bp of the 5'-noncoding region and the 312 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, the poly(A) tail was also found. A potential ribosome-binding site was located 30 nucleotides upstream to the initiation codon in the 5'-noncoding region. The amino acid sequence deduced from the nucleotide sequence was 433 amino acids in length and showed very high homology (82%) to the amino acid sequence of gamma gamma enolase (neuron-specific enolase: NSE), although the nucleotide sequence showed slightly lower homology (75%). The size of NNE mRNA was approximately 1800 bases by Northern transfer analysis and much shorter than that of NSE mRNA (2400 bases) indicating a short 3'-noncoding region. A dot-blot hybridization and Northern transfer analysis of cytoplasmic RNA from the developing rat brains using a labeled 3'-noncoding region of cDNA (no homology between NSE and NNE) showed a decrease of NNE mRNA at around 10 postnatal days and then a gradual increase to adult age without changes of mRNA size. Liver mRNA did not show any significant change during development.     

Content of neuron-specific and non-neuron-specific enolase isoenzymes in human brain structures

The distribution of neuron-specific (NSE) and non-neuronal (NNE) isoforms of glycolytic enzyme enolase (EC 4.2.I.II.) in the human brain was studied using immunoenzyme assay. The maximum NSE concentration was measured in the frontal and occipital brain cortex, hippocampus, limbic cortex and hypothalamus (12-14 micrograms/mg of water-soluble protein), the minimum level was observed in the brain stem structures (3-6 micrograms/mg). The maximum NNE content was determined in thalamus (34 micrograms/mg). The data can prove useful for the study of enolase isoform distribution in the brain of neurological and psychiatric patients.     

Neuroprotection by α-Lipoic Acid in Streptozotocin-Induced Diabetes

Glial cells provide structural and metabolic support for neurons, and these cells become reactive to any insult to the central nervous system. The streptozotocin (STZ) rat model was used to study glial reactivity and the prevention of gliosis by alpha-lipoic acid (alpha-LA) administration. The expression of glial fibrillary acidic protein (GFAP), S100B protein, and neuron specific enolase (NSE) was determined as well as lipid peroxidation (LPO) and glutathione (GSH) levels in some brain tissues. Western blot analyses showed GFAP, S100B, and NSE levels significantly increased under STZ-induced diabetes in brain, and LPO level increased as well. Administration of alpha-LA reduced the expression both of glial and neuronal markers. In addition, alpha-LA significantly prevented the increase in LPO levels found in diabetic rats. GSH levels were increased by the administration of alpha-LA. This study suggests that alpha-LA prevents neural injury by inhibiting oxidative stress and suppressing reactive gliosis.     

Isolation and characterization of non-neuronal enolase (NNE) from Neurospora crassa and comparison with neuron specific enolase isolated from neuroblastoma cell line NG108

Enolase is a vital enzyme of the glycolytic pathway. It exists mainly in two forms, non-neuronal enolase (NNE) and neuron specific enolase (NSE). Neurospora crassa, a filamentous fungus, was used as the source of pure NNE, and by using DEAE-cellulose and a Sephadex G-150 column chromatography highly purified enzyme (20.4 fold purification with 54.7 percent recovery) was obtained. The development profile of the enzyme shows a peak value after 90 hours of mycelial growth from conidia of N. crassa. In this respect, it differs from neuroblastoma NSE where the peak value of the enzyme activity appears 7 1/2 hours after the splitting of the cells. N. crassa enolase (NNE) is more thermolabile than NG108 NSE and N. crassa enolase is more sensitive to urea, chloride, and fluorophosphate. The Km values for 2-phosphoglycerate and Mg++ were 0.34 mM and 0.47 mM, respectively, for N. crassa enolase, whereas these values were 1.1 mM and 3.1 mM, respectively, in the case of neuroblastoma NSE. N. crassa enolase is a dimer molecule of molecular weight 85,000 daltons. N. crassa enolase is not neutralized by NSE antisera and neutralized by NNE antisera as opposed to neuroblastoma NSE.     

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.     

Targeting Enolase in Reducing Secondary Damage in Acute Spinal Cord Injury in Rats

Spinal cord injury (SCI) is a complex debilitating condition leading to permanent life-long neurological deficits. The complexity of SCI suggests that a concerted multi-targeted therapeutic approach is warranted to optimally improve function. Damage to spinal cord is complicated by an increased detrimental response from secondary injury factors mediated by activated glial cells and infiltrating macrophages. While elevation of enolase especially neuron specific enolase (NSE) in glial and neuronal cells is believed to trigger inflammatory cascades in acute SCI, alteration of NSE and its subsequent effects in acute SCI remains unknown. This study measured NSE expression levels and key inflammatory mediators after acute SCI and investigated the role of ENOblock, a novel small molecule inhibitor of enolase, in a male Sprague–Dawley (SD) rat SCI model. Serum NSE levels as well as cytokines/chemokines and metabolic factors were evaluated in injured animals following treatment with vehicle alone or ENOblock using Discovery assay. Spinal cord samples were also analyzed for NSE and MMPs 2 and 9 as well as glial markers by Western blotting. The results indicated a significant decrease in serum inflammatory cytokines/chemokines and NSE, alterations of metabolic factors and expression of MMPs in spinal cord tissues after treatment with ENOblock (100 µg/kg, twice). These results support the hypothesis that activation of glial cells and inflammation status can be modulated by regulation of NSE expression and activity. Analysis of SCI tissue samples by immunohistochemistry confirmed that ENOblock decreased gliosis which may have occurred through reduction of elevated NSE in rats. Overall, elevation of NSE is deleterious as it promotes extracellular degradation and production of inflammatory cytokines/chemokines and metabolic factors which activates glia and damages neurons. Thus, reduction of NSE by ENOblock may have potential therapeutic implications in acute SCI.     

人骨髓间充质干细胞在成年大鼠脑内的迁移及分化

骨髓间充质干细胞 (mesenchymalstemcells,MSCs)是目前备受关注的一类具有多向分化潜能的组织干细胞 ,体外可以分化为骨、软骨、脂肪等多种细胞。因此 ,MSCs是细胞治疗和基因治疗的种子细胞之一。为了探索MSCs的迁移和分化趋势 ,为帕金森病 (Parkinsondisease,PD)的干细胞治疗提供理论和实验依据 ,本实验将体外扩增并转染增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)的人骨髓MSCs注入PD大鼠脑内纹状体 ,观察了人骨髓MSCs在大鼠脑内的存活、迁移、分化以及注射MSCs前后大鼠的行为变化。结果表明 ,人骨髓MSCs在大鼠脑内可存活较长时间 ( 10周以上 ) ;随着时间的延长 ,MSCs迁移范围扩大 ,分布于纹状体、胼胝体、皮质以及脑内血管壁 ;免疫组化法检测证实MSCs在大鼠脑内表达人神经丝蛋白 (neurofilament,NF)、神经元特异性烯醇化酶 (neuron specificeno lase,NSE)以及胶质原纤维酸性蛋白 ( glialfibrillaryacidprotein ,GFAP) ;PD大鼠的异常行为有所缓解 ,转圈数由 8 86±2 0 9r/min下降到 4 87± 2 0 6r/min ,统计学分析P <0 0 5为差异显著。以上观察结果表明 ,骨髓MSCs有望成为治疗PD的种子细胞     

Modifications in energy metabolism during the development of chick glial cells and neurons in culture

Developmental changes in lactate dehydrogenase (LDH), enolase, hexokinase (HK), malate dehydrogenase (MDH), and glutamate dehydrogenase (GDH) activities were measured in cultures of pure neurons and glial cells prepared from brains of chick embryos (8 day-old for neurons, 14 day-old for glial cells) as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain extracts during the development of the nervous tissue in the chick embryo and during the post-hatching period. A significant increase of MDH, GDH, LDH, and enolase activities are observed in neurons between 3 and 6 days of culture, whereas simultaneously a decrease of HK values occurs. In the embryonic brain between 11 and 14 days of incubation, which would correspond for the neuronal cultures to day 3 through 6, modifications of MDH, GDH, HK, and enolase levels are similar to those observed in neurons in culture. Only the increase of LDH activity is less pronounced in vivo than in cultivated cells. The evolution of the tested enzymatic activities in the brain of the chick during the period between 7 days before and 10 days after hatching is quite similar to that observed in cultivated glial cells (prepared from 14 day-old embryos) between 6 and 18 days of culture. All tested activities increased in comparable proportions. The modifications of the enzymatic profile indicate that some maturation phenomena affecting energy metabolism of neuronal and glial elements in culture, are quite similar to those occuring in the total nervous tissue. A relationship between the development of the energy metabolism of the brain and differentiation processes affecting neuroblasts and the glial-forming cells is discussed.     

Release of neuron specific enolase (NSE) in cerebrospinal fluid following experimental lesions of the rat brain

Neuron-specific enolase (NSE) levels were measured by sandwich enzymo-immunoassay as well as by enzymatic assay in rat cerebrospinal fluid (CSF), following mechanical lesions of the brain tissue. Significant increases of NSE were observed in CSF, with a peak 2 h following lesions located near the lateral ventricle. Values returned to normal around 48 h later. In another experimental group, lesions were realized further away from the lateral ventricle; the elevation of NSE in CSF reached the maximal value 11 h later. In addition, measurements which were performed following lesions at the same location but of various sizes, indicated that the quantity of NSE released is proportional to the extent of brain damage. The possible factors which govern the time course and amount of NSE release in CSF are discussed. These results suggest that NSE could be a useful and easily detected marker of neuronal damage.     

Antibodies to neurofilament protein and other brain proteins reveal the innervation of peripheral organs

Summary Monoclonal and polyclonal antibodies to neurofilament proteins, neuron-specific enolase, glial fibrillary acidic protein and S-100 have been used to demonstrate nerves, ganglion cells and the supportive glial system of the innervation of various organs. The female genitalia, the urinary tract, the respiratory system, the pancreas, the heart and the skin of several mammalian species, including rat, mouse, guinea pig, cat, pig, monkey and man were fixed in parabenzoquinone and portions of each organ were snap frozen. Serial or free-floating thick cryostat sections were stained using indirect immunofluorescence and peroxidase anti-peroxidase immunocytochemistry. In addition, the newly described and highly sensitive immunogold-silver staining technique was used on Bouin's-fixed and wax-embedded tissues.Antibodies to neurofilament proteins seemed to react with neuronal structures in all the species studied. Alternately stained serial sections showed a similar distribution of neurofilament proteins and neuron-specific enolase-containing nerves. Neuron-specific enolase staining had a diffuse appearance and was found to be highly variable, indicating that the neuron-specific enolase content might be related to the physiological state of the nerves and ganglion cells, whereas antibodies to neurofilament protein gave a consistently intense and very clear picture of the ganglion cells and nerve fibres. Antibodies to S-100 stained supportive elements of the peripheral nervous system in all tissues examined, whereas antibodies to glial fibrillary acidic protein were more selective.Abbreviations GFAP glial fibrillary acidic protein - NSE neuron-specific enolase - PBS phosphate-buffered saline - PAP peroxidase anti-peroxidase - FITC fluorescein-isothiocyanate     

Combined immunostaining of neurofilaments, neuron specific enolase, GFAP and S-100. A possible means for assessing the morphological and functional status of the enteric nervous system

Neurofilaments, part of the cytoskeletal network, and neuron specific enolase, a major enzyme in glycolysis, are both present in central and peripheral neurons. Glial fibrillary acidic protein and S-100, on the other hand, are soluble proteins which are found exclusively in the supportive cells of the nervous system, i.e. the glial cells. Examination was made, using immunocytochemistry, of all main areas of the gastrointestinal tract of three mammalian species, rat, pig and man. By applying serial tissue sectioning, it was possible to study the relative occurrences of the two neuronal markers in the same cell bodies and to examine the relationships of the neurons with the glial cells as revealed by the antibodies to glial fibrillary acidic protein and S-100. Both neurofilaments and neuron specific enolase were localised to an extensive system of enteric nerves, with the level of neuron specific enolase-immunoreactivity showing greater variability than that observed using antibodies to neurofilaments. Comparison of the occurrence of neuron specific enolase and neurofilament immunoreactivity in serially sectioned neuronal cell bodies revealed that a minor population stained only with antibodies to neurofilaments. The equivocal or absent neuron specific enolase-immunoreactivity in some perikarya may reflect variations in functional status within the nervous system. Glial fibrillary acidic protein- and S-100-immunoreactivities were confined to glial cells which, in this normal tissue, were always in close association with the neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative alterations of S-100 protein and neuron specific enolase in the rat nervous system after chronic 2,5-hexanedione exposure

The regional changes in quantities of the glial S-100 protein and the neuron specific enolase in the rat nervous system have been studied after long-term exposure to 2,5-hexanedione. The wet weights of most of the examined nervous tissues were found to be reduced, with an extensive effect seen in the brain stem. Using dot immunobinding assays, the concentrations of S-100 were found to be increased in most of the examined tissues, but unaffected in the brain stem. The total amount of S-100 per tissue was markedly reduced in the brain stem. The content of neuron specific enolase was reduced only in the brain stem. Thus the effects of 2,5-hexanedione on the nervous system varied regionally. The brain stem was severely atrophied with a reduction of neuronal as well as of glial marker proteins. Other brain regions contained increased glial cell marker proteins as signs of progressive astroglial reactions.     

Effect of ethanol on adenosine triphosphatase and enolase activities in rat brain and in cultured nerve cells

The effect of alcohol on enzymes involved in energy metabolism of nervous tissue were analyzed, in vivo after acute and chronic ethanol administration to rats and in vitro by addition of 50 mM and 100 mM ethanol to the medium of cultured nerve cells: chick neurons, chick glial cells, a neuronal cell line (MT17) and a glial tumoral cell line (C6). The parameters we measured were (Na⁺,K⁺), Mg²⁺ and ecto Ca²⁺,Mg²⁺ ATPase activities involved in transport phenomena and enolase activities (non neuronal NNE and neuron specific enolase NSE) as markers of nerve cell maturation. In vivo, after chronic ethanol administration (Na⁺,K⁺) ATPase activity was increased while Mg²⁺ dependent activity was not affected. Enolase activity was decreased. Acute ethanol administration decreased (Na⁺,K⁺) ATPase activity, while Mg²⁺ dependent activity was not affected. In cultured nerve cells ethanol effect was dose, time and cell type dependent; alterations of the cell membrane by trypsinization of the tissue before seeding modifies the effect of ethanol on the enzymes we analyzed. Our results suggest that alcohol effect on nerve cells depends mainly on the lipoprotein structure of the cell membranes which may have different properties from one cell type to another.