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This study was designed to investigate whether fractional anisotropy (FA) contributes to study pathologic changes in SCI. Here rats were divided into a control group and three injury groups. Those in the injury groups were administered a mild, moderate, and severe contusion injury at the T10 vertebral level, respectively. Three rats were randomly selected from each group at 6, 24 and 72 h after SCI for imaging examination. Magnetic resonance diffusion tensor imaging was FA and tractography. Once magnetic resonance was completed, blood was collected and serum levels of neuron specific enolase (NSE) and soluble protein-100β (S-100β) were determined. Then animals were sacrificed and histopathologic examination was conducted. The spinal cord in the SCI model rats produced prominent tissue damages characterized by neuronal injury in the affected regions. An obvious decrease in FA happened 24 h after SCI, and at 72 h, FA tended to be stable. There were significant differences in the serum levels of NSE and S-100β between 6 h and 24 h, respectively. FA significantly related with the serum testing results at 24 h. FA may be used as a marker for different severities of SCI. The optimal time for examination is at 24 h post-injury in rat.

α-Synuclein is a neuronal protein implicated in the etiology of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Whilst increased α-synuclein expression due to gene duplication or triplication can cause familial PD, previous studies of α-synuclein levels in idiopathic disease have produced conflicting data. We quantified α-synuclein mRNA and soluble protein in five human post-mortem brain regions from four groups of individuals with PD, DLB, Alzheimer's disease (AD) and matched controls. α-Synuclein mRNA levels, measured using quantitative real-time PCR, did not differ significantly between groups in any brain regions examined. In contrast, levels of soluble α-synuclein protein, measured by ELISA, were significantly lower in 4 of the 5 regions for patients with DLB, and in 2 of the 5 regions for patients with PD, compared to controls. Soluble α-synuclein protein levels were not significantly different in the AD patients, compared to controls, in 4 of the 5 regions. This study indicates that although levels of soluble α-synuclein protein are lower in DLB and PD, there is no evidence for a corresponding decrease in α-synuclein mRNA levels. This might result from altered translation, or removal of α-synuclein protein from a soluble detectable state, either by turnover or conversion to an insoluble form.

The present study investigated cerebrospinal fluid (CSF) biomarkers for estimating degeneration of the central nervous system (CNS) in experimental dogs with GM1 gangliosidosis and preliminarily evaluated the efficacy of long-term glucocorticoid therapy for GM1 gangliosidosis using the biomarkers identified here. GM1 gangliosidosis, a lysosomal storage disease that affects the brain and multiple systemic organs, is due to an autosomal recessively inherited deficiency of acid beta-galactosidase activity. Pathogenesis of GM1 gangliosidosis may include neuronal apoptosis and abnormal axoplasmic transport and inflammatory response, which are perhaps consequent to massive neuronal storage of GM1 ganglioside. In the present study, we assessed some possible CSF biomarkers, such as GM1 ganglioside, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), neuron-specific enolase (NSE) and myelin basic protein (MBP). Periodic studies demonstrated that GM1 ganglioside concentration, activities of AST and LDH, and concentrations of NSE and MBP in CSF were significantly higher in dogs with GM1 gangliosidosis than those in control dogs, and their changes were well related with the months of age and clinical course. In conclusion, GM1 ganglioside, AST, LDH, NSE and MBP could be utilized as CSF biomarkers showing CNS degeneration in dogs with GM1 gangliosidosis to evaluate the efficacy of novel therapies proposed for this disease. In addition, we preliminarily treated an affected dog with long-term oral administration of prednisolone and evaluated the efficacy of this therapeutic trial using CSF biomarkers determined in the present study. However, this treatment did not change either the clinical course or the CSF biomarkers of the affected dog, suggesting that glucocorticoid therapy would not be effective for treating GM1 gangliosidosis.

The current study examines nestin expression after intracerebral hemorrhage (ICH), the role of different blood components in nestin upregulation, and the possibility that low doses of thrombin that induce tolerance to brain injury (thrombin preconditioning) might also induce nestin expression. Adult male Sprague-Dawley rats received an intracaudate injection of either whole blood, thrombin (1 or 5 U) or red blood cells (RBCs). Animals were sacrificed for single and double labeling immunohistochemistry to identify which cells express nestin, and for Western blotting to quantify nestin expression. By immunohistochemistry, nestin immunoreactivity was present in large numbers of astrocytes, surrounding the hematoma from day 3 to 1 week after ICH. After 2 weeks, nestin immunoreactivity was co-localized with a neuronal marker (neuronal specific enolase). By Western blot analysis, nestin was strongly expressed at day 3 (P0.01) and 1 week (P0.01), and expression persisted for at least 1 month (P0.05). Intracerebral injection of thrombin or lysed RBCs resulted in a marked increase in nestin expression. Interestingly, injection of a low dose of thrombin that induces brain tolerance also upregulated nestin. The ICH-induced nestin expression in astrocytes may reflect an early response of these cells to injury, while the delayed expression in neurons might be a part of the adaptative response to injury perhaps leading to recovery of function. Nestin induction by a low dose of thrombin suggests that specific receptor-mediated pathways are involved in inducing nestin expression and that nestin may play a role in thrombin preconditioning.

We have established a cell line (TB) from a cerebrospinal fluid (CSF) specimen of a patient with a primary leptomeningeal melanomatosis. TB cell line was immunoreactive with the antibodies for low molecular weight neurofilament protein, vimentin, neuron-specific enolase, chromogranin, synaptophysin and HMB-45 (an antibody sensitive and specific for melanoma). When TB cells were transplanted into nude mice, the same immunohistochemical pattern present in cultured cells was found but surprisingly, a positive staining for desmin was observed. Significant amounts of serotonin and its metabolite were detectable. Retinoic acid but not nerve growth factor was able to induce differentiation towards a neuronal phenotype. In summary, TB cells represent primitive neuroectodermal cells having the potential for neuronal, myoblastic and possibly melanoblastic differentiation.

Blocks of fresh tissue from embryonic rat cerebral cortex, dissociated either prior to or after freezing, were stored for up to 10 months in liquid nitrogen at − 196°C with 7% dimethylsulfoxide (DMSO) as cryoprotectant. Slow freezing and rapid thawing, a reduced medium volume and moderate elevation of both extracellular K + (20 mM) and sera (20%) promoted survival of neurons (up to 7 weeks) on polylysine-coated glass coverslips. Although there was cell loss associated with freezing, the surviving cells developed morphological and immunocytochemical properties similar to those expressed by unfrozen cells when using anti-GFAP (glial fibrillary acidic protein), anti-NSE (neuron specific enolase) and anti-GABA (gamma-aminobutyric acid) antibodies. A comparative computer-aided analysis of the morphometric patterns of GABA-IR neurons allowed the individualization of two similar populations in both fresh and frozen controls. NSE- and GABA-immunoreactive (IR) cells were counted in frozen controls and thienyl-phencyclidine (TCP)-treated cultures. The latter yielded fewer cells whereas it was the opposite in fresh TCP-treated cultures. In view of these findings, a detailed analysis of the effects of both neuroprotective and neurotoxic agents on frozen cells in undertaken before considering that cryopreservation might be a suitable storage method for clinical trials of neuron grafting.

Protein kinase C (PKC) is involved in neural plasticity. The phosphorylation of the myristoylated alanine-rich protein kinase C substrate (MARCKS) in the left intermediate and medial hyperstriatum ventrale (IMHV) of the chick brain has been shown previously to correlate significantly with the strength of learning in filial imprinting. The distribution of PKC alpha, beta I, beta II and PKC gamma in the brain of 1-day-old dark-reared chicks was determined immunocytochemically, using the monoclonal antibodies MC5 and 36G9, raised against purified PKC alpha beta and PKC gamma, respectively. PKC gamma-stained cells were distributed widely in the telencephalon, including all hyperstriatal structures (including the IMHV), the hippocampus, neostriatum, ectostriatum and archistriatum. There were fewer stained cells in the septum and the least cellular staining was in the paleostriatum primitivum. Fluorescent double-labelling with neuron-specific enolase (NSE) and with the glial calcium-binding protein S100 suggested that PKC gamma immunoreactivity was present in neurones but not in glia. The distribution of PKC alpha beta-stained cells was more limited, with staining in the archistriatum, hippocampus and septum but not in the hyperstriatum. However, there was PKC alpha beta-staining of some fibres in the IMHV (but little elsewhere in the hyperstriatum ventrale), in the neostriatum, paleostriatal complex and the lobus parolfactorius. Double-labelling with NSE and S100 revealed PKC alpha beta/S100-positive glial cells present in the paleostriatal region only. There was some PKC alpha beta-staining of putative neurones in the hippocampus, septum and archistriatum. The differential distribution of PKC isoenzymes suggests that in the IMHV some axonal inputs contain PKC alpha beta whereas some postsynaptic cells contain the gamma form of PKC.

Two brain-specific proteins, S-100beta and neuron-specific enolase (NSE), are released systemically after cerebral lesions, but S-100beta levels sometimes rise in the absence of neuronal damage. We hypothesized that S-100beta is a marker of blood-brain barrier (BBB) leakage rather than of neuronal damage. We measured both proteins in the plasma of patients undergoing iatrogenic BBB disruption with mannitol, followed by chemotherapy. Serum S-100beta increased significantly after mannitol infusion (P

Neuron-specific enolase (NSE) is an isoform of the glycolytic enzyme, enolase, and is found in neurons and neuroendocrine cells. We evaluated cerebral immunohistologic and plasma changes in NSE in rats from 2 h to 15 days following permanent or transient middle cerebral artery occlusion (MCAO). At 1-2 days post-MCAO, loss of NSE immunofluorescence from within neurons to the extracellular space was observed in the infarcted areas of all MCAO animals. NSE also was identified intravascularly throughout the brain following MCAO. NSE in plasma was determined by a specific radioimmunoassay. Plasma NSE following permanent or transient MCAO was increased significantly from that observed in controls (2.8 +/- 0.3 ng/ml) beginning at 2 h and persisting for 2.5 days post-MCAO (maximum levels of 8.8 +/- 0.9 to 9.6 +/- 0.5 ng/ml after 6-12 h; P < 0.05, n = 4-9). Quantified contralateral forelimb and hindlimb neurological deficits in these animals were significant and persisted for at least 15 days following MCAO but were not observed following sham surgery. These data suggest that MCAO-induced cortical infarction and neurological dysfunction is associated with neuronal depletion and vascular redistribution of brain NSE resulting in a measurable increase in plasma NSE. Such diffusion of NSE into the cerebral vasculature and systemic circulation from ischemic tissue can be expected to serve as a marker for the incidence of cerebral damage in acute and chronic ischemic brain infarcts.

We have developed a sensitive enzymatic-immunoassay to quantify the level of gamma-enolase (a specific neuronal enzyme) which is released from cultured cells after exposure to various toxins. We show that this method can estimate selectively neuronal cell death without significantly interfering with glial cell death. Indeed, no gamma-enolase is released when glial cells are killed with free-radical producing agents. Experiments comparing the levels of neuronal cell death induced by NMDA or free-radical producing drugs, performed either by measuring gamma-enolase release or using the classical fluorescein diacetate method, yielded similar results. In addition to selectively follow neuronal death in a mixed population of neurons and glial cells, this method provides a way of determining the cell death kinetics from a single culture dish, since enolase can be measured on small samples taken from the culture medium. Finally, we propose these two methods as being complementary and useful neuronal and other cellular death indexes and also to understand the complex problem of glial influence on neuronal survival or death.

Sprague-Dawley dams were treated with 3 v/v % ethanol in liquid diet from the 8th day gestation through 3 weeks nursing period. Offsprings, ages 1, 2 and 3 weeks, and their dams were studied. Brain weights and their total proteins were not affected by this ethanol treatment. Total enolase activity/mg protein and its isoenzymes, i.e. non-neuron-specific, hybrid and neuron-specific, (mumol/min/mg protein) in 100,000 g supernate, were significantly lower in the treated. Additionally, enolase isoenzyme transformation was delayed.

Dissociated cell cultures were prepared from human spinal cords of 7-10-week-old fetuses. After 10 weeks progressive neuronal necrosis was observed in controls whereas N/1-(2-thienyl)cyclohexyl/piperidine (TCP) enhanced the survival time of the cells. After 21 weeks the number of gamma-aminobutyric acid (GABA)ergic and neuron specific enolase (NSE)-stained neurons was higher in the TCP-treated cultures than in controls. TCP appears to be a promising drug for long term survival of neurons.

The study employed an immunochemical quantification of brain cell marker proteins in addition to quantitative morphology in order to provide a more multifacetted and characterized model for an excitotoxic CNS lesion. The importance of the approach in the evaluation of the potential of neuroprotective agents is emphasized. The S-100 protein, the glial fibrillary acidic (GFA) protein, neuron specific enolase (NSE) and neuronal intermediary filament polypeptides (NF 68 and NF 200) were measured with a dot-immunobinding assay, 3-30 days after a unilateral injection of N-methyl-D-aspartate (NMDA) in the left dorsal hippocampus of the rat. After 3 days, the neuronal cell loss averaged 80% in the hippocampus. The S-100 content was reduced 3 days after injection, but was 150% of control at 30 days. GFA increased constantly from days 3 to 30. The neuronal marker proteins were all markedly reduced 7 days after injection. However, at 30 days, NF 68 and NF 200 were close to control (80%). Increasing content would reflect regeneration and sprouting of neurites. The content of the neuronal cytoplasmic marker, NSE, was significantly lower than control also at 10 and 30 days, although a gradual recovery could be traced.

Transgenic mice expressing heme oxygenase-1 (HO-1) using the neuron-specific enolase promoter were impaired in learning the Morris water maze compared to nontransgenic littermates. The memory of the HO-1 mice for the location of the platform was similarly impaired when tested using a probe trial after 7 training blocks, but performance on visible platform trials was similar for both groups of mice. Importantly, both HO-1 and nontransgenic mice had normal sensorimotor function, and performed the same on a Y-maze alternation task, highlighting the specificity of memory deficit in the spatial navigation task. These results suggest that carbon monoxide, one product of HO-1 activity, interferes in the development of spatial navigation memory, and may play a role in normal memory function.

Statoacoustic ganglion (SAG) cells were grown in primary culture and the appearance of different neuronal phenotypes was investigated. Analysis criteria were shape, size and staining for the immunocytochemical markers: neurofilament proteins (NF-200 kDa), neuron-specific enolase (NSE), calretinin, a calcium-binding protein and substance P, a neurotransmitter. Cultures were prepared from dissociated SAG cells of 13 gestation-day-old mouse embryos. Neurons were identified and counted after 7 days in vitro. At this stage, neurons were organized in small clusters forming an extensive network of neurites grown on a layer of fibroblasts and glia. Most neurons identified by NF or NSE immunoreactivity showed a typical adult-like bipolar profile. The diameters of the neurons were between 5.62 and 17.00 microns and displayed a normal distribution (mean: 10.6 microns). Two distinct subpopulations were identified by the expression of calretinin and substance P. Calretinin-immunoreactive neurons were large and very rare and had a mean diameter of 11.3 microns; the distribution of substance P was more extensive than that of calretinin and identified a population of small neurons with a mean diameter of 8.9 microns. The distributions of these two markers in SAG cultures were consistent with in vivo results. In conclusion, dissociated SAG cell cultures appear to be a suitable model for analyzing the development of the immunocytochemical and functional characteristics of the neurons of this inner ear ganglion.

The role of alcohols in affecting neuromorphogenesis was investigated in a single cell type, pheochromocytoma (PC12). The effect of ethanol at physiological concentrations in this system leads to enhanced morphological and functional differentiation in combination with nerve growth factor (NGF). PC12 cells treated with a suboptimal concentration of NGF (30 ng/ml) and an alcohol (87 mM) underwent rapid morphological differentiation which was dependent upon the side chain length of the alcohol MeOH less than EtOH less than PrOH less than BuOH. Pyrazole at either 5 or 10 mM had no effect on alcohol induced neurite extension. Assessment of the degree of differentiation promoted by the various alcohols was quantified by an increase in neurite extension, a decrease in the incorporation of [3H]thymidine, an increase in acetylcholine esterase (AChE) activity and immunostaining with neuron specific enolase. Thus, alcohols may function in a specific manner by interacting with transmembrane signalling pathways which promote gene expression and neuronal differentiation.

An immunoblot procedure was developed to quantify the amount of tryptophan hydroxylase (TpOH), the rate limiting enzyme in the synthesis of serotonin, in the rat raphe dorsalis nucleus (NRD). Using this method we have studied the time course variations in TpOH protein level after a single p-chlorophenylalanine (PCPA) i.p. injection (300 mg/kg). PCPA provoked a rapid and large decrease of TpOH in the NRD, without affecting neuron-specific enolase in the NRD or TpOH in the locus coeruleus. The decrease in TpOH was maximum (-60% of the control value) 2 days after the drug administration and followed a monoexponential law which allowed us to estimate the half-life of this enzymatic protein as 1.43 days and to postulate that, during these 2 days, TpOH synthesis was inhibited. The neosynthesis of TpOH molecules from 2 to 7 days was estimated to be 57.8 U TpOH/NRD/day which was comparable to the initial steady state of synthesis (48.44 U TpOH/NRD/day). In vivo administration of 6-fluorotryptophan or in vitro incubation of raphe homogenates with either halogenated derivative had no effect on TpOH protein levels. PCPA should be an interesting tool to study the turnover rate of TpOH protein.

PGP 9.5 is a new cytoplasmic neuron-specific protein structurally and immunologically distinct from neuron-specific enolase. A specific antiserum has now shown that this protein is widely distributed in vertebrate brains and is also present in cells of the human diffuse neuroendocrine system. PGP 9.5 appears to be older than neuron-specific enolase in evolutionary terms, as an immunologically-related protein can be found in species as remote as the trout.

gamma-Enolase has been believed to be distributed only in the neurons and it was frequently labelled as neuron-specific enolase. However, recent precise studies have suggested a wider distribution of the protein. It can also be found in neuroendocrine cells, some mesodermal tissues, and some malignant tumors originating from tissues without the antigen in a normal condition. In early rat embryos, before the formation of neural tissues, a sensitive immunoassay system revealed a substantial amount of gamma-enolase, though it is not yet clear where the antigen is located. In the present study, tissue distribution of gamma-enolase in early human embryos was studied using an immunohistochemical method and it was suggested that the protein is present not only in neural tissue primordium but also in most tissues in the youngest embryo of 6.3 mm crown-rump length. Many of the immunoreactive non-neural tissues, however, lost immunoreactivity with the advancement of the embryonic stage while neural tissues became more intensely stained. In the embryo of 24 mm length, the staining pattern was almost the same as that of reported adult men. In embryonic tissues such as notochord and mesonephros, which disappear in the due course of growth, the antigen was also found. These findings will suggest that the antigen is rather common in undifferentiated tissues but then localizes in neural elements with advancing age. Our findings may be useful in explaining why early rat embryos, before the formation of neural tissues, showed a considerable amount of gamma-enolase and why many undifferentiated tumors, originating from tissues which did not have the antigen in normal condition, revealed the antigen.

The cellular localization of alpha-, beta- and gamma-subunits of enolase isoenzyme was studied immunohistochemically in various human brain tumors, retinoblastomas and pituitary adenomas (total, 91 cases). The alpha-subunit was found in almost all brain tumors except in medulloblastoma cases. A case of medullomyoblastoma contained beta-subunit-positive myoblastic cells, which was also positive for human myoglobin. The gamma-subunit was frequently observed in medulloblastomas, retinoblastomas and pituitary adenomas. However, gamma-subunit was also present in some non-neuronal brain tumors such as astrocytomas and oligodendrogliomas. Enzyme immunoassay technique also confirmed the presence of gamma-subunit in a medulloblastoma and two retinoblastomas.

The in vitro effects of the neurotoxic compounds, acrylamide and 2,5-hexanedione, on several glycolytic enzymes including enolase, phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactic dehydrogenase (LDH) were studied in rat brain. A differential sensitivity of the enzymes to the inhibitory effects of the neurotoxins was observed. The order of increasing sensitivity to 2,5-hexanedione was enolase -- GAPDH -- PFK and to acrylamide the order was PFK -- enolase -- GAPDH. Neither neurotoxin inhibited LDH. The inhibition of enolase by acrylamide exhibited a mixed type pattern in double reciprocal plots. The inhibition could be completely reversed by dialysis indicating that it did not involve covalent bond formation. In the presence of dithiothreitol (DTT) or glutathione the inhibition of enolase by either acrylamide or 2,5-hexanedione was potentiated. Activity of enolase inhibited by both acrylamide and DTT could not be restored to pre-inhibition rates following dialysis indicating that an irreversible interaction between acrylamide and enolase had taken place. The results suggest that neurotoxic compounds which produce distal axonopathies have a common pattern of attack on glycolytic enzymes and that interruption of glycolysis is the underlying biochemical basis for both the physiological and morphological damage caused by these compounds.

In Alzheimer's disease there is a reported loss of large cells in the cholinergic nucleus basalis of Meynert. It has been suggested, however, that there may be neurons in the nucleus basalis in Alzheimer's disease which are atrophied and therefore difficult to distinguish from neuroglia by size. This has important therapeutic implications and we have attempted to clarify the situation using a neuron-specific antiserum directed against neuron-specific enolase (NSE). Sections of nucleus basalis were stained using this antiserum and the neuronal cross-sectional area was measured. A profile of neuronal distribution with area was obtained, by image analysis, and compared in controls and patients with Alzheimer's disease. A significant 29% overall loss of neurons was found in Alzheimer's disease with a much greater loss (61%) of large neurons and concurrent increase (59%) in small neurons. Analysis of variance showed significant reduction in mean cross-sectional neuronal area as a consequence of this shift in frequency towards a preponderance of small cells. It is suggested that in the nucleus basalis in Alzheimer's disease, large neurons are not completely lost; many are shrunken and thus excluded from the previous studies of large cells counted in Nissl-stained material. That there is partial preservation of these neurons makes it more likely that cholinergic dysfunction, characteristic of Alzheimer's disease, will be amenable to neurotrophic influence.

The presence of the two forms of enolase, neuron-specific enolase (NSE) and non-neuronal enolase (NNE), have been examined in biopsy material of human neuroblastoma, ganglioneuroblastoma, ganglioneuroma and cultured neuroblastoma cells, after separation with ion exchange chromatography. The enolase activities were inhibited in the presence of NaCl but remained active in KCl, which were used in the chromatographic step. The relative NSE levels in the neuroblastoma tissues were found to be lower than in the histopathologically more differentiated forms of the tumour, i.e. ganglioneuroblastoma and ganglioneuroma. The human neuroblastoma in vitro cell lines SK-N-SH, SH-SY5Y, SK-N-MC and IMR-32 contained considerably lower relative levels of NSE compared to the levels in the neuroblastoma biopsies. After treatment of the cultures cells with nerve growth factor or dibutyryl-cAMP some cells showed morphological differentiation and concomitantly an increase in the NSE levels. The results indicate that NSE might be useful as a marker for differentiation in human neuroblastoma.

The immunocytochemical localization for hydroxyindole O-methyltransferase (HIOMT), neuron-specific enolase (NSE) and S-100 protein in the bovine pineal gland is described in this paper. Most of the pinealocytes showed immunoreactivities to a HIOMT antiserum and an NSE antiserum, simultaneously. Neither HIOMT-nor NSE-immunoreactivity was observed in a few pinealocytes. On the other hand, dispersed interstitial cells (glial cells) were stained only by an S-100 protein antiserum. No evident HIOMT-immunoreactivity was observed in the retina and intestinal mucosa, where HIOMT has been suggested to occur.

The distribution of 3 forms of rat enolase (alpha alpha, alpha gamma and gamma gamma forms), including nervous system-specific forms (alpha gamma and gamma gamma), was determined in various tissues with a sensitive enzyme immunoassay system. The brain and spinal cord contained more than 100 pmol/mg protein of the alpha alpha, alpha gamma and gamma gamma enolases. Organs such as the lungs, heart, spleen, liver, and kidney contained similarly high levels of alpha alpha enolase, but these tissues contained alpha gamma and gamma gamma enolases at levels less than 1% of the central nervous tissues. High levels of the alpha gamma (greater than 10 pmol/mg) and the gamma gamma (greater than 1.5 pmol/mg) forms were found in rectum, bladder, and uterus. In gut, major portions of the nervous system-specific forms were localized in the muscle layers. Skeletal muscle and diaphragm, which are composed of striated muscle, contained low levels of 3 forms of enolase. Megakaryocytes separated from the suspension of bone marrow contained 11.3, and 0.53 amol/cell of the alpha alpha and gamma gamma enolases, respectively, with little, if any, of the alpha gamma form.

The spectrum of enalose enzyme forms has been examined in several lines of neuroblastoma cells and compared to those present in whole brain. The neuron specific enolase (NSP) is greatly decreased in the cultured cells as judged by activity profiles and radioimmunoassay. The synthesis of neuronal enolase appears to be extensively depressed in these cells while the total enolase activity is not affected. The non-neuron form of enolase (NNE) apparently compensates for the lack of the neuronal forms in the cultured cells. The preponderance of NNE in cultured neurons suggests that this enzyme is present in immature neurons, and that neurobla cells are not fully differentiated with respect to the enolase function. Dibutyryl cyclic adenosine monophosphate treatment does increase NSP levels in mouse neuroblastoma cells, but not to the levels expected for fully differentiated neurons. The results indicate that NSP is a molecular correlate of fully differentiated neurons.

Three forms of the glycolytic enzyme, enolase [2-phospho-D-glycerate hydrolase (E.C. No. 4.2.1.11)] have been prepared from rat whole brain extract. The most acidic enolase form is neuron specific enolase (NSE) which had previously been designated neuron specific protein (NSP). The least acidic form designated non-neuronal enolase (NNE) has been purified and compared structurally, immunologically and functionally to NSE. NNE is a dimer of 86,500 M.W. consistint of two very similar subunits. The data establish that NNE is larger than NSE which has been shown to be composed of two apparently identical 39,000 molecular weight subunits (78,000). NNE is less acidic than NSE having a pI of 5.9 compared to the value of 4.7 for NSE. Structural and immunological analysis establishes that the NNE subunit is distinct from the NSE subunit, and are therfore products of two separate genes. The structural designation of NSE is (gammagamma) and that of NNE (alpha' alpha'). NSE is strictly localized in neurons indicating that the gene coding for the gamma subunit is only expressed in neuronal cells. The intermediate brain enolase form has been partially purified; structural and immunological evidence indicate that it is a hybrid molecule consisting of one NNE subunit and one NSE subunit (alpha'gamma).

The enolase (EC 4.2.1.11) isoenzymes, neuron-specific enolase (NSE, gamma gamma) and non-neuronal enolase (NNE, alpha alpha), are markers for neurons and glia, respectively, in adult mammalian brain. In developing fetal and early postnatal brain, levels of non-neuronal enolase (NNE) are high. Neuron-specific enolase (NSE) appears only after neurogenesis begins in a given region and only slowly attains adult levels. Immunocytochemistry in developing rat and rhesus monkey brain reveals that proliferative zones that give rise to neurons are NNE(+). Thus, nerve cells must undergo a switch from NNE to NSE. In addition, study of neurons in cerebellum and neocortex reveals that they are NNE(+) during migration and only become NSE(+) in their final location, presumably after making full synaptic connections. Such migrating cells may contain hybrid enolase (alpha gamma) and some (e.g. cerebellar stellate/basket cells) may not completely switch over to NSE even in the adult. Neuron-specific enolase is not only a specific molecular marker for mature nerve cells, but is closely correlated to the differentiated state.

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.

A technique is described for a two-cell co-cultivation system which permits in vitro evaluation of neuron-glia interactions. Primary astroglial enriched cultures from newborn rat cerebral hemispheres, striatum or cerebral cortex, grown for 3 days, were co-cultivated with primary neuron-containing cultures from 15- to 17-day rat embryo cerebral hemispheres, substantia nigra or brainstem, respectively, grown for 10 days on polysine-coated surfaces. The neuronal cells were identified morphologically and immunohistochemically by antibodies to neuron-specific enolase. The two cultures were grown together for 7 days, separated by a U-formed 1 mm glassrod. The results show that neurons exert a morphogenetic effect on astroglial cells in the form of extension of cell processes. The co-culture system allows investigation of potent local humoral interactions between astroglial cells and neurons.

The cellular localization of the two forms of monoamine oxidase (MAO A and MAO B) was studied by measuring their activities in rat striatum following unilateral stereotaxic injection of kainic acid to produce selective degeneration of striatal neurons and subsequent proliferation of astrocytes. The results demonstrated a persistent loss of 15–20% in MAO A activity, whereas MAO B activity decreased initially by 25% and then increased to more than twice the control value by 54 days after lesions. The changes in activity were compared to parallel estimates of the postsynaptic neuronal enzyme markers glutamic acid decar☐ylase (GAD) and neuron-specific enolase (NSE), astroglial enzyme markers glutamine synthetase (GS) and non-neuronal enolase (NNE), and the presynaptic enzyme marker DOPA decar☐ylase (DDC). The results suggest that a small amount of striatal MAO A is present in kainic acid-sensitive postsynaptic striatal neurons and that MAO B is probably localized in both neurons and astrocytes.

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) form homologous sets of multiple molecular forms. The central nervous system of mammals contains mostly tetramers (G 4 ) and monomers (G 1 ). Their proportions have been shown to vary during maturation in rat brain. In order to examine whether a similar evolution occurs in the human, we performed parallel studies of the activity, solubility and molecular forms of acetylcholinesterase in rat and human brains at various stages. We find both similarities and differences: in rat brain, the enzyme increases mostly postnatally but in human brain acetylcholinesterase reaches a maximum at birth. There is an increase in the proportion of G 4 and a decrease in the solubility of this form in the absence of detergent in human as well as in rat brain. These changes occur around birth in rat, but during early pregnancy, before 11 weeks in human brain. In both species, the solubility of the enzyme in detergent-free buffers decreases progressively from more than 50% before birth to about 10–20% in the adult. In addition we analyzed butyrylcholinesterase as well as the levels of the neuron-specific enolase and of the glial S-100 protein. In human, γγ-enolase rises to its adult level after birth, but before the S-100 protein.

A rapid technique for separating and quantitating the three enolase isozymes present in rodent brain and sciatic nerve was developed using high-pressure liquid anion-exchange chromatography. At pH 7.9, one cationic and two anionic enzyme forms were separated with baseline resolution in an imidazole buffer containing ethylenediaminetetraacetic acid (EDTA) and magnesium. The recovery of enolase activity was 90% or greater for brain and 85% for sciatic nerve. Chromatography of liver and axon-free (degenerated) sciatic nerve allowed the identification of non-neuronal, hybrid, and neuron-specific enolase isozymes. These enzyme forms, respectively, constituted 40%, 29% and 19% of total activity in brain, and 63%, 13% and 4% of total activity in normal sciatic nerve.

Cultured human SH-SY5Y neuroblastoma cells could be induced to differentiate morphologically and biochemically followed by growth inhibition, by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The cells showed a limited differentiation when treated with substances known to increase the intracellular concentration of cyclic AMP. When these substances were combined with TPA, morphological differentiation and growth inhibition of the cells were potentiated. In contrast, these substances inhibited the TPA-induced increase in noradrenaline concentration and the relative activity of neuron-specific enolase. Both the intracellular concentration of cyclic AMP and the cytosolic level of cyclic AMP-binding components were similar in control and TPA-treated cells. It is suggested that cyclic AMP has a limited and non-regulatory role in the initiation of differentiation of SH-SY5Y cells. The effect of cyclic AMP is probably coupled mainly to the polymerization of microtubules, thus enhancing the morphological differentiation of the cells.

Neuron-specific enolase (NSE) is an isoenzyme of the glycolytic enzyme enolase (EC 4.2.1.11) which is 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 stains or strictly morphologic criteria for identifying nerve cells.

Butyrate treatment results in the rapid formation of processes on F98 anaplastic glioma cells. The morphological differentiation occurs more rapidly and to a greater extent than that induced by nerve growth factor (NGF). The incorporation of [3H]thymidine is virtually stopped 1 day after treatment of F98 cells with sodium butyrate. Butyrate also caused a large increase in ornithine decarboxylase activity in F98 cells between 6 and 12 h after treatment. This was inhibited by both cycloheximide and actinomycin-D. NGF did not cause these effects. Butyrate also caused an increase in neuron-specific enolase (NSE) content in F98 cells. This effect appeared to be specific for butyrate. Since NSE induction is characteristic only of neurons and neuroendocrine cells, this finding indicates that butyrate is capable of inducing biochemical differentiation along neuronal lines in undifferentiated glioma cells.

The possible rapid retrograde axonal transport of neuron-specific enolase was investigated employing a sensitive radioimmunoassay. The right sciatic nerve of rats was ligated and 6 and 24 h later the nerve was cut in two 5.0 mm segments immediately proximal and distal to the ligature. Similar were taken from the contralateral nerve as a control. No increase in the amount of neuron-specific enolase in the right-distal segment was observed indicating that the protein is not a component of retrograde axonal transport.

Prominent variations in the concentration and composition of poly (A)-containing messenger RNA were found to occur during the postnatal development of the rat cerebellum. The concentration of mRNA (μg/g cerebellar tissue) was determined to be the highest on the tenth postnatal day, at the onset of synaptogenesis. Short nonabundant mRNA chains continuously increase in amount during cerebellar development, while the fraction of long translatable mRNAs decreases. The overall ability of cerebellar mRNA to stimulate the incorporation of [ 35 S]methionine into polypeptides in reticulocyte lysate apparently does not change. The proteins synthesized in vitro by cerebellar mRNA from different developmental stages were therefore analyzed by single and 2-dimensional gel electrophoresis. The diversity of these proteins and the levels of many of them were found to vary with cerebellar development. The newly synthesized brain forms of enolase and creatine kinase were identified by their migration coordinates in the 2-dimensional protein gels, and increase in the abundance of their directing mRNAs was found to accompany the differentiation of cerebellar interneurons. The extent of modification in cerebellar mRNA was determined to be much higher than the consequent changes in the composition of cerebellar proteins. We propose to use the ontogenetic variations in the levels of specific cerebellar mRNA species in normal and malformed cerebellum to identify proteins specific to particular types of cerebellar neurons.

The function in neurons of the vitamin D-dependent calcium ion-binding-protein of 28 kDa mol. wt., calbindin D28K, is unknown. In order to find a simple system for studying the function of this protein, neuron-rich primary cultures derived from brains of 16-day-old rat embryos were analyzed for the presence of calbindin D28K by immunocytochemical mono- and double-labelling techniques. The studies were carried out between the 5th and 23rd day after seeding. In contrast to the neuronal marker neuron-specific enolase which was found in nearly all cells in the culture, calbindin D28K was expressed only in a subpopulation of neurons. Calbindin D28K-positive cells were intensely stained in their cell bodies and were also stained in their processes. Astroglial cells identified by the presence of the processes. Astroglial cells identified by the presence of the specific marker glial fibrillary acidic protein did not express calbindin D28K. Therefore, calbindin D28K is a useful marker for defining a neuronal subpopulation in neuron-rich primary cultures. Such cultures may be employed as a tool in searching for function(s) of calbindin D28K.

Neuron-specific enolase (NSE) has been localized only in neurons and cells with characteristics of neurons. The immunocytochemical localization of NSE was examined in guinea pig cochleae to determine if hair cells, which have some neuronal characteristics, would show NSE-like immunoreactive labeling. NSE-like immunoreactivity was seen in inner hair cells but not in outer hair cells. This is the first report of NSE-like immunoreactivity in a receptor cell. NSE-like immunoreactivity was also seen in efferent fibers and terminals and in both type I and type II spiral ganglion cells. The finding of NSE-like immunoreactivity in inner but not outer cells adds to the number of differences found between them and may be related to differences in function and action.

Rat brain contains 3 forms of enolase, a neuron-specific form (NSE), a hybrid form, and a non-neuronal form (NNE) which were separated by DEAE-cellulose column chromatography. The enolase activity corresponding to each form of the enzyme eluted from the columns was determined spectrophotometrically. Using this assay procedure, an activity ratio (%NNE/%NSE) was calculated for cerebellum, brainstem, sciatic nerve, adrenals and liver. The results indicated excellent agreement between this enzymatically determined ratio and published values of a similar ratio (NNE/NSE) determined by radioimmunoassay for enzyme protein. Following in vivo destruction of neurons by intracerebral injection of the selective neurotoxin, kainic acid, there was a significant decrease in the activity of NSE and hybrid enolase (neuronal forms) and no change in the activity of NNE (glial form). These data indicate that separation and measurement of enolase species is useful to determine levels of these species in normal tissue and to estimate neuronal damage biochemically in brain lesions.

Developmental changes of the neuron-specific enolase (NSE) and the non-neuronal enolase (NNE) were determined in pure neuronal, or mixed cultures of neuronal and glial cells, prepared from brains of 8-day-old chick embryos, as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain during the development of the nervous tissue in the embryo and during the post-hatching period. In the whole brain, the total enolase activity increases progressively until the adult level, the NSE appears only 4 days before hatching. The total enolase activity of pure neurons increases until 8 days of culture but the NSE never appears during this period. Mixed cultures of neurons and glial cells present an increase of total activity until 14 days of culture and neuronspecific form of the enzyme can be observed after 6 days of culture. Our results suggest that NSE appears only in the functional brain and is expressed in cultivated neurons only when glial cells are present simultaneously.

The effect of acrylamide on selected glycolytic and citric acid cycle enzymes has been studied in denervated cat sciatic nerves in vitro and in vivo. The enzyme activity of glyceraldehyde-3-phosphate dehydrogenase (GADPH), neuron specific enolase (NSE), succinate dehydrogenase (SDH), and lactate dehydrogenase (LDH), has been examined in saline-perfused, desheathed and denervated peroneal (P) and tibial (T) nerves from cats treated with acrylamide (15 mg/kg/day, s.c.) or vehicle for 15 days. GAPDH activity in Schwann cells in denerevated P and T nerve stumps was 2.0- and 2.3-fold higher than normal P and T nerve values. GAPDH activity in Schwann cells in denervated P and T nerves of acrylamide-treated cats was markedly reduced (56% and 61% of untreated denervated nerves, respectively). LDH and SDH activities were unaffected by acrylamide and NSE activity was absent in denervated nerve stumps. Acrylamide (0.5 and 20 mM) inhibited GAPDH activity in denervated nerve homogenates by 67% and 29%, respectively. This study demonstrates that acrylamide inhibits GAPDH in Schwann cells. The significance of GAPDH inhibition by acrylamide in denervated nerves and its relation to distal axonopathy has been discussed.

The effect of monomeric acrylamide, a potent neurotoxic agent, on total and neuron specific enolase activity was studied in vitro and in vivo. Acrylamide (10 mM) completely inhibited total enolase activity of rat brain soluble fractions. The I50 concentration was 3.7 mM. In rats chronically treated with acrylamide (550 mg/kg total) and exhibiting marked symptoms of neurotoxicity, neuron specific enolase activity was not detectable in sciatic nerves and was only 60% of control activity in brain. Total enolase activity in both central and peripheral nervous tissues was unchanged from control. The results suggest that inhibition of neuron specific enolase may be an important factor in the development of acrylamide neuropathy by interfering with glycolysis in neuronal tissue.

Neurons grown in cultures of dissociated brain cells degenerate when exposed to anoxia and deprived of glucose. We have developed culture systems in which neurons can be grown in the presence or absence of astrocytes and have used them to study the influence of astrocytes on the neuronal degeneration induced by anoxia and glucopenia. Cultures were prepared from fetal rat forebrains. Mixed cultures contained neurons (identified by immunocytochemical staining of neuron-specific enolase, NSE) and about an equal number of non-neuronal cells (identified by glial fibrillary acid protein). Pure neuronal cultures were prepared by adding a cytostatic compound (cytosine arabinoside) to the medium. Treated cultures were exposed for 4 h to glucose-free medium and an atmosphere of 95% N2 and 5% CO2, whereas control cultures were left in the usual medium containing glucose and in an atmosphere composed of 95% air and 5% CO2. After an interval of 24 h, cultures were fixed, taken for NSE staining, and the number of surviving neurons was counted. Exposure to anoxia and glucopenia reduced the number of surviving neurons in pure neuronal cultures to 5–10% of control levels. In contrast, in mixed cultures 40–60% of the neurons survived these conditions. Anoxia without glucose deprivation reduced the number of surviving neurons in both types of cultures to the same extent as anoxia combined with glucopenia. Glucose deprivation alone was ineffective. The findings suggest a protective influence of astrocytes on neurons under anoxic conditions. γ- d -Glutamylglycine protected neurons in both types of cultures from anoxia-induced degeneration. However, no evidence was obtained for an involvement of excitatory amino acids in the protective influence of astrocytes.