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PubMed1989

Tubulin isotype usage in vivo: a unique spatial distribution of the minor neuronal-specific beta-tubulin isotype in pheochromocytoma cells.

The neuronal cells of vertebrates express two beta-tubulin isotypes, called Class II and Class III, that are neuronal specific. In order to determine the distribution of the minor Class III isotype, site-directed antibodies were raised to synthetic peptides representing the carboxyl terminal, isotype-defining domains of the tubulins. These antibodies were applied to PC12 cells at various stages of differentiation. The Class III isotype was found to be expressed in undifferentiated PC12 cells as well as in cells at every stage of differentiation. The concentration of the Class III isotype, relative to the total beta-tubulin complement, did not change significantly. Indirect double immunofluorescence microscopy demonstrated that the Class III isotype was found in the soma and the neurites of differentiated PC12 cells; this spatial pattern of Class III expression paralleled the total beta-tubulin pattern. Although the anti-Class III antiserum could stain in vitro assembled neuronal microtubules in a filamentous pattern, a close examination of the Class III staining pattern in flattened PC12 cells revealed that this isotype was not incorporated into the nonaxoplasmic array of microtubules. Rather, the Class III isotype was localized in a nonfilamentous, granular pattern that was not readily extracted with nonionic detergent. Cells treated with taxol and then flattened and stained showed that the Class III isotype could be induced to assemble into microtubule bundles by taxol. Thus, the minor neuronal beta-tubulin isotype appears to be spatially specialized in its pattern of expression.

Adrenal Gland Neoplasms↗
PubMed1990

Detection of neuron specific enolase concentrations in cerebrospinal fluid from patients with neurological disorders by means of a sensitive enzyme immunoassay.

An enzyme linked immunosorbent assay (ELISA) for the detection of neuron specific enolase (NSE) in cerebrospinal fluid (CSF) was developed. The sensitivity of the ELISA was less than 1 microgram/ml. This sensitivity is comparable with radioimmunoassays which have the disadvantage that radiolabelled products are used. The developed assay was used to measure cerebrospinal fluid neuron specific enolase (CSF-NSE) levels in 1178 patients with neurological disorders to establish its potential usefulness and clinical application. CSF-NSE levels in this group of patients were independent of sex and no correlation with age was found. CSF-NSE was significantly increased in Creutzfeldt-Jacob disease, meningeal hemorrhage, thrombosis, Guillain-Barré syndrome and in schizophrenia.

Animals↗
PubMed1990

The structure of the human gene encoding protein gene product 9.5 (PGP9.5), a neuron-specific ubiquitin C-terminal hydrolase.

Database search using a bovine thymus ubiquitin C-terminal hydrolase sequence indicated 54% sequence identity with the abundant human neuron-specific protein gene product 9.5 (PGP9.5), which was then shown to possess the same activity [Wilkinson, Lee, Deshpande, Duerksen-Hughes, Boss & Pohl (1989) Science 246, 670-673]. A yeast counterpart of the enzyme is also known. The human PGP9.5 gene, described here, spans 10 kb, contains nine exons and displays 5' features some common to many genes and some common with neurofilament neuron-specific enolase and Thy-1-antigen gene 5' regions.

Amino Acid Sequence↗
PubMed1991

Neuron-specific enolase concentrations in serum in benign liver diseases.

Neuron-specific enolase (EC 4.2.1.11; NSE), a glycolytic enzyme produced by neuronal and neuroendocrine cells, is used as a tumor marker of neuroendocrine cancers, especially small-cell lung cancer. We have evaluated the behavior of NSE in nonmalignant liver diseases in 161 patients who underwent a thorough clinical and biochemical evaluation. Nine of the 161 patients (5.6%)--three of the 86 cirrhotic patients (3.5%), and six of the 75 noncirrhotics (8%)--had abnormal concentrations of NSE. Of the numerous clinical and analytical factors considered, none correlated significantly with NSE. The low false-positive rate, similar to or even lower than for other benign diseases, and the absence of association with the numerous characteristics of liver diseases that we studied, support the lack of any substantial metabolism of NSE by the liver. Consequently, the coexistence of a benign liver disease does not limit the usefulness of this enzyme as a tumor marker.

Adolescent↗
PubMed1997

A neuron-specific splicing switch mediated by an array of pre-mRNA repressor sites: evidence of a regulatory role for the polypyrimidine tract binding protein and a brain-specific PTB counterpart.

Tissue- and stage-specific alternative splicing events are widespread in mammals, yet the factors and mechanisms that direct these important posttranscriptional events are poorly understood. In this study, we focus on the 24-nt exon of the GABA(A) receptor gamma2 pre-mRNA, which is subject to neuron-specific and developmental splicing regulation in the rat cerebellum. Here we show biochemical evidence for a mechanism that directs the selective repression of the neuron-specific exon in non-neuronal splicing extracts derived from HeLa cells. Key evidence includes the discovery that the pathway of gamma2 pre-mRNA splicing switches from exon skipping to exon selection in splicing reactions with a short RNA competitor containing the 3' splice site region upstream of the 24-nt exon. In this assay, exon selection results from the coordinate activation of both flanking introns. A detailed dissection of this pre-mRNA region shows that it contains four repressor sites clustered around the branch site and extending into the 24-nt exon. These repressor sites are pyrimidine rich and bind avidly to the polypyrimidine tract binding protein (PTB) in HeLa nuclear extracts as determined by UV crosslinking/competition assays. Repression of the exon selection pathway is closely associated with the appearance of a specific RNA-protein complex, indicative of an inhibitor complex, that assembles on the repressor array. Upon the switch to the exon selection pathway, a substantial decrease in the inhibitor complex and a reciprocal increase in spliceosome complex A is observed. Excess recombinant PTB squelches the splicing switch and reestablishes exon skipping as the predominant splicing pathway. Extracts prepared from rat brain nuclei show reduced levels of conventional PTB compared to other splicing factors. Nonetheless, the rat brain nuclear extracts contain an activity that assembles an analogous inhibitor complex efficiently. We report a 59-kDa protein, p59, which has an electrophoretic mobility distinct from HeLa and rat kidney PTB, and which behaves in RNA binding assays as if it is the PTB counterpart in rat brain. Evidence that rat brain p59 is structurally related to PTB stems from western blot and immunoprecipitation analysis with a monoclonal antibody specific for the hnRNP I isoform of PTB. A model describing how the repressor array directs coordinate splicing regulation of flanking introns in the context of overlapping positive regulatory elements is discussed. The sequence, (5') UUCUCU (3'), in a pyrimidine context is associated with one class of intron splicing repressor sites that binds PTB in a variety of pre-mRNAs that are regulated by tissue-specific programs.

Alternative Splicing↗
PubMed2001

Neuron-specific PGP9.5 expression in rat hair follicle development and cycle.

Protein gene product PGP9.5 is a neuron-specific ubiquitin C-terminal hydrolase. We found that it also has immunoreactivity in the hair follicle of the Wistar rat dorsal skin and its expression patterns change with the development and cycle. During the morphogenesis, the PGP9.5 was expressed in the hair germ and hair peg elongated from the epidermis, and became restricted in the outer root sheath as the development progressed. In catagen, however, the PGP9.5 was detected in the tailing epithelial strand of the regressing proximal follicle epithelium, and in the keratinocytes directly contacted with the club hair, but rarely in the outer root sheath. With the beginning of the anagen of the second hair follicle, the PGP9.5 was again expressed in the second hair germ, and in the keratinocytes surrounding the remaining club hair and of distal follicle of the first hair. These findings showed that PGP9.5 is not specific to the neuron but is also involved in the hair follicle, and should provide new insight into the development and regression of the hair follicle.

Animals↗
PubMed1990

Monoclonal antibodies against sensory neuron specific antigens define the extent of neuronal abnormality in the mf mutant rat.

The mutant rat mutilated foot (mf) is affected by a sensory neuropathy which does not involve the parts of the body innervated by the thoracic cord. The possibility that sensory cells subserving clinically normal regions may be functionally spared by the mutation has been investigated by studying the expression of cell surface oligosaccharides by dorsal root ganglia (DRG) and their central processes in the spinal cord. The study included 3 lactoseries epitopes (TC6, LD2 and LA4) and the globoseries epitope SSEA3. The results show that at cervical and lumbar levels in mf rats there are reduced numbers of DRG cells reacting with the various antibodies and less immunostaining in the dorsal horns. The unexpected finding that thoracic ganglia and cord share similar appearances suggests that, in spite of being normal in number and able to produce normal amounts of substance P, thoracic DRG cells in mf rats take part in the mutation as shown by their inability to produce normal amounts of oligosaccharides and to transport them to the axon terminals.

Animals↗
PubMed1986

Secretion of neuron-specific enolase, prolactin, growth hormone, luteinising hormone and follicle stimulating hormone by "functionless" and endocrine-active pituitary tumours in vitro.

Secretion of the neuroendocrine marker neuron-specific enolase by 24 pituitary tumours was measured in maintenance tissue culture. Eleven endocrine-active and 13 "functionless" tumours were defined by measurement of prolactin, growth hormone, luteinising hormone (LH) and follicle stimulating hormone (FSH) secretion rates in vitro and the corresponding plasma hormone levels. Measurement of prolactin secretion provided a clear distinction between true prolactinomas and "functionless" tumours causing hyperprolactinaemia by stalk compression (pseudoprolactinomas). A previous report of LH and/or FSH secretion by the majority of "functionless" tumours was confirmed, but plasma levels of LH and FSH were usually normal. It is argued that LH and FSH are not the major hormones secreted by "functionless" tumours. A high production rate of neuron-specific enolase appears to be characteristic of the cell type from which most "functionless" tumours derive.

Adenoma↗
PubMed1985

[Serum neuron-specific enolase as a marker of lung cancers].

Neuron-specific enolase (NSE) in sera of lung cancer patients was studied in order to evaluate its clinical significance as a tumor marker. The subjects included 15 normal volunteers, 13 cases without malignant neoplasms or neuronal diseases and 42 lung cancer cases. NSE was quantified by a double antibody radioimmunoassay. As one of the sera from normal volunteers and control patients showed an NSE content 10 ng/ml or more, values of 10 ng/ml or over were considered to be positive. Seventeen of 42 sera from lung cancer patients showed positive NSE levels. Histological evaluation revealed that the degrees of NSE positiveness for small cell carcinoma, large cell carcinoma, squamous cell carcinoma and adenocarcinoma were 73%, 50%, 33%, and 21%, respectively, and that all the positive cases except for one were confined to disease stages III or IV. The level of NSE in patients with 10 ng/ml or more before surgery decreased to within normal limits 1-2 weeks after surgery Localization of NSE could be confirmed immunohistologically in small cell carcinoma cells. In conclusion, NSE was considered to be very useful as a tumor marker of the lung, especially in small cell carcinoma for diagnosis and determination of disease extent and response to therapy, and also in non-small cell carcinoma for the evaluation of treatment effectiveness.

Adenocarcinoma↗
PubMed1986

Cerebrospinal fluid neuron-specific enolase is reduced in Alzheimer's disease.

Neuron-specific enolase (NSE), a glycolytic enzyme enolase found in brain, was examined in the cerebrospinal fluid and serum of 30 patients with presumptive Alzheimer's disease (AD) and of 13 healthy controls and evaluated as a measure of neuronal functional activity associated with AD. The cerebrospinal fluid NSE levels of patients with AD were significantly reduced and serum NSE levels were significantly increased from controls. Cerebrospinal fluid NSE levels may be representative of central nervous system cell loss or a decrease in neuronal functional activity associated with AD.

Aged↗
PubMed1985

A comparison of the evolutionary distribution of the two neuroendocrine markers, neurone-specific enolase and protein gene product 9.5.

One- and two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting has been used to examine the phylogenetic distribution of the two neuronal and neuroendocrine proteins, neurone-specific enolase and protein gene product 9.5, in animal brains. A new immunoblotting procedure was used in which complex two-dimensional patterns of brain proteins were transferred to nitrocellulose paper simultaneously with the Coomassie Blue stain. This produced a copy of the blue spot pattern against which brown protein spots reacting in a specific antibody-immunoperoxidase procedure could be identified unequivocally. Extracts of human, bovine, sheep, rabbit, rat, guinea-pig, chicken, trout, and frog brains were examined. Proteins cross-reacting with antisera to the human forms of both proteins could be demonstrated in all species examined. This suggests that proteins corresponding to neurone-specific enolase and protein gene product 9.5 could have evolved at least 400 million years ago and have been highly conserved throughout evolution.

Animals↗
PubMed2003

Prediction of early clinical severity and extent of neuronal damage in anterior-circulation infarction using the initial serum neuron-specific enolase level.

CONTEXT: Prompt and precise measurement of neuronal damage in acute cerebral infarction is important to determine the prognosis of functional outcome. A feasible biochemical marker such as the neuron-specific enolase (NSE) level has been used to detect various diseases involving the central nervous system. OBJECTIVE: To determine whether the initial serum NSE level is a useful marker for predicting the severity of clinical neurological deficits and the extent of neuronal damage in acute anterior-circulation infarction. DESIGN: Case-control study with biochemical-clinicoradiological correlation. SETTING: Tertiary care center. PARTICIPANTS: Eighty-one patients and 77 age- and sex-matched control subjects. MAIN OUTCOME MEASURES: Patients with anterior-circulation infarction underwent intravenous serum NSE sampling within 24 hours after symptom onset. Recent infarction was confirmed by T2-weighted and diffusion-weighted magnetic resonance imaging of the brain about 1 week after the onset of stroke. Volumetric analysis of infarction was also performed. The National Institutes of Health Stroke Scale score was measured on admission to the hospital and 1 week after symptom onset. RESULTS: The patients' initial serum NSE levels were statistically significantly higher than the controls (P<.05). The initial serum NSE level highly correlated with the volume of infarction seen on T2-weighted magnetic resonance imaging of the brain (r = 0.62, P<.001) and with the National Institutes of Health Stroke Scale score obtained on hospital admission (r = 0.42, P =.002) and on the seventh day after the onset of stroke (r = 0.44, P<.001). CONCLUSION: The initial serum NSE level is a reliable predictor for the extent of neuronal damage and the severity of clinical neurological deficits in acute anterior-circulation infarction.

Aged↗
PubMed1996

Cyclin-dependent kinase 5 (Cdk5) and neuron-specific Cdk5 activators.

While cyclin-dependent kinase 5 (Cdk5) is widely distributed in mammalian tissues and in cultured cell lines, Cdk5-associated kinase activity has been demonstrated only in mammalian brains. An active form of Cdk5, called neuronal cdc2-like kinase (Nclk) has been purified from mammalian brain and shown to be a heterodimer of Cdk5 and a 25 kDa protein, which is derived proteolytically from a 35 kDa brain and neuron-specific protein. The protein is essential for the kinase activity of Cdk5 and is therefore designated neuronal Cdk5 activator, p25/35Nck5a. Nclk appears to have important neuronal functions. The changes in Cdk5 and Nck5a expression appear to correlate with the terminal differentiation of neurons of the mouse embryonic brain. Transfection of cultured cortical neurons with dominant negative cdk5 mutants or Nck5a antisense DNA may reduce neurite growth, suggesting that Nclk plays an active role in neuron differentiation. A number of cytoskeletal proteins including neurofilament proteins, the neuron-specific microtubule associated protein tau, and the actin binding protein caldesmon are in vitro substrates of Nclk. Although Nck5a has cyclin-like activity, it shows minimal amino acid sequence identity to members of cyclin family proteins. The mechanism of activation of Cdk5 by Nck5a differs from that of cyclin activation of Cdks in that full Cdk5 kinase activity can be achieved in the absence of phosphorylation of Cdk5. An isoform of Nck5a, a 39 kDa protein has been cloned and shown to share extensive amino acid identity and the mechanism of Cdk5 activation with Nck5a. These proteins may represent a subfamily of Cdk activators distinct from cyclins.

Amino Acid Sequence↗
PubMed1993

Transcriptional activity of the neuron-specific enolase (NSE) promoter in murine embryonic stem (ES) cells and preimplantation embryos.

Mouse embryonic stem (ES) cells were transfected with a plasmid composed of an E. coli lacZ gene fused to 1.8 kb of rat neuron-specific enolase (NSE) promoter sequences. While this reporter construct had been shown previously to function exclusively in postmitotic neurons and neuro-endocrine cells of transgenic mice, stably transfected ES cell clones unexpectedly displayed beta-galactosidase (beta-Gal) activity in the undifferentiated state. This transcriptional activity of the heterologous NSE promoter was confirmed by the identification of endogenous NSE mRNA in undifferentiated ES cells, mouse morulae and blastocysts. NSE protein, however, could not be found in undifferentiated ES cells. Interestingly, in ES cells which were cultured for 7 days under differentiation conditions in vitro, beta-Gal activity decreased to basal levels consistent with the parallel down-regulation of endogenous NSE mRNA. In contrast, prolonged culture of ES cells under differentiation conditions led to the reappearance of NSE mRNA and beta-Gal activity after 17 days. Significant increases in beta-Gal activity were also observed in ES cells which were cultured either on dishes coated with attachment factors such as laminin and gelatin or in the presence of nerve growth factor (NGF). These results suggest that i) transcriptional control mechanisms regulating neuronal gene expression are present at early developmental stages in the mouse and ii) ES cells provide a useful in vitro model system for the analysis of developmentally regulated cellular and molecular events coupled to neuron-specific enolase promoter activity.

Animals↗
PubMed1980

Expression of neuronal specificities in "transdifferentiating" cultures of neural retina.

Cells dissociated from the neural retina of embryonic chick differentiate into lens and pigment cells, when cultured in vitro. Using 3.5-day-old and 8.5-day-old chick embryos, we examined whether neuronal specificities would be expressed in such transdifferentiating cultures of neural retinal cells. The synthesis of acetylcholine and gamma-aminobutyric acid (GABA) and the activity of choline acetyl transferase (CAT) was searched for in these cultures. The synthesis of an appreciable amount of these two putative neurotransmitters was detected in cultures of 3.5-day-old embryonic retinas by about 15 days. The activity of CAT was maximum in 7-day cultures of the 3.5-day-old materials and in 2-day cultures of the 8.5-day-old materials, and then decreased. Concomitant with the decrease of CAT-activity, delta-crystallin became detectable and increased thereafter. CAT-activity changed in parallel with the increase in the number of small neuroblast-like cells in cultures. The results demonstrate that the neuronal specificity identified by the appearance of acetylcholine and GABA and of the enzyme for the synthesis of acetylcholine is expressed in the early period of transdifferentiating cultures, which would later differentiate into lens and pigment cells. The possible mechanisms of the transition from neuronal to non-neuroretinal specificities of the transdifferentiating cultures are discussed.

Acetylcholine↗
PubMed2000

A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing.

The Nova paraneoplastic antigens are neuron-specific RNA binding proteins that participate in the control of alternative splicing. We have used the yeast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-binding protein (PTB). The expression of this protein, brPTB, is enriched in the brain, where it is expressed in glia and neurons. brPTB interacts with Nova proteins in cell lines and colocalizes with Nova within neuronal nuclei. We previously found that Nova binds to a pyrimidine-rich RNA element present upstream of an alternatively spliced exon, E3A, in glycine receptor alpha2 (GlyRalpha2) pre-mRNA, and this binding is implicated in Nova-dependent regulation of splicing. Cotransfection assays with a GlyRalpha2 minigene demonstrate that brPTB antagonizes the action of Nova to increase utilization of GlyRalpha2 E3A. brPTB binds to a 90-nt GlyRalpha2 RNA adjacent to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova. When a putative binding site for brPTB on the GlyRalpha2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dependent exon selection disappears. These results suggest that brPTB is a tissue-restricted RNA binding protein that interacts with and inhibits the ability of Nova to activate exon selection in neurons.

Alternative Splicing↗
PubMed2004

Ontogenetic observations on the vomeronasal organ in two species of tamarins using neuron-specific beta-tubulin III.

Callitrichid primates (tamarins, marmosets) have extreme variation in the vomeronasal organ (VNO), including ontogenetic differences in the neuroepithelium and vomeronasal duct (VND) patency at birth. Such differences render the timing and extent of VNO maturation debatable in callitrichids, but no studies have used neuron-specific immunohistochemical markers to address this question. The present study compared the number of VNO epithelial cells that express immunoreactivity to neuron-specific beta-tubulin III (BT), VNO length, and VNO cross-sectional area between two species of tamarins (Leontopithecus rosalia and Saguinus geoffroyi) that differed in perinatal VND patency. Neonatal lemurs and adult marmosets and bushbabies were also examined for a comparison to species previously shown to have a relatively large amount of VNO neuroepithelium and patent VNDs. The head of each specimen was serially sectioned in the coronal plane. Based on known rostrocaudal start/stop points of the VNO, selected unstained sections were used for BT protocols and area measurement at three percentiles (25th, 50th, 75th) in each specimen. Each section was photographed and enlarged for cell counts and measurement of cross-sectional epithelial area. In each specimen, the number of BT(+) cells in the VNO was counted at each percentile and expressed as a number per mm(2). Results indicated that lemur VNOs had a dense population of BT(+) cells at birth, but the VNO was more varied in the tamarin species. S. geoffroyi had few or no BT(+) cells in VNOs of neonates, which had fused VNDs, but had an increased BT(+) population by 1 and 2 months postnatal age, when the VND was patent. Of the species with patent VNDs at birth, neonatal L. rosalia had a denser population of BT(+) cells compared to S. geoffroyi, though not to the degree seen in neonatal lemurs or adult marmosets and bushbabies. These findings show that BT immunohistochemistry is a useful comparative method for the study of VNOs in subadult primates. Since the quantity of nonsensory VNO epithelium varies substantially between species, epithelial area measurements may be misleading, and BT(+) cell counts appeared to be the best quantitative method for comparing receptor neuron numbers among primates. It is suggested that the greater BT(+) cell population in L. rosalia at all subadult stages examined reveals an earlier maturation of the neuroepithelium compared to S. geoffroyi. Further investigation should consider whether this may relate to a comparatively brief subadult ontogeny and early onset of adult behaviors in L. rosalia compared to other tamarins studied to date.

Age Factors↗