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.     

创伤后应激障碍大鼠海马与前额叶皮质中OREXIN受体的失调

目的:从食欲素(Orexin, ORX)系统挖掘创伤后应激障碍的神经生物学机制。方法:以单次延长刺激(single prolonged stimulation,SPS)法复制创伤后应激障碍(post traumatic stress disorder,PTSD)大鼠模型,以行为学结合血清中皮质酮和神经元特异性烯醇化酶进行模型评价,通过酶联免疫吸附试验(Elisa)分析大鼠血清与脑脊液中OREXIN水平,以实时荧光定量聚合酶链反应(RT-PCR)检测海马与前额叶皮质中的Orexin受体基因表达。结果:实验成功的复制了PTSD模型,与对照组相比,模型组大鼠血清中皮质酮和神经元特异性烯醇化酶的含量均极显著升高(P0.01),脑脊液中Orexin A、Orexin B的含量均显著升高(P0.01),海马和皮质中ORX1R与ORX2R均极显著下降(P0.01)。结论:PTSD大鼠的应激损伤与Orexin及其受体表达水平的变化密切相关。     

Fatty acid binding protein as a serum marker for the early diagnosis of stroke: a pilot study

No biological marker is currently available for the routine diagnosis of stroke. The aim of this pilot study was to determine whether heart-fatty acid binding protein (H-FABP) could be used as a valid diagnostic biomarker for stroke, as compared with neuron-specific enolase (NSE) and S100B proteins. Using two-dimensional gel electrophoresis separation of cerebrospinal fluid proteins and mass spectrometry techniques, FABP was found elevated in the cerebrospinal fluid of deceased patients, used as a model of massive brain damage. Because H-FABP, a FABP form present in many organs, is also localized in the brain, an enzyme-linked immunosorbant assay was developed to detect H-FABP in stroke versus control plasma samples. However, H-FABP being also a marker of acute myocardial infarction (AMI), troponin-I and creatine kinase-MB levels were assayed at the same time in order to exclude any concomitant heart damage. NSE and S100B levels were assayed simultaneously. These assays were assessed in serial plasma samples from 22 control patients with no AMI or stroke, 20 patients with AMI but no stroke, and 22 patients with an acute stroke but no AMI. Twenty-two out of the 22 control patients and 15 out of the 22 stroke patients were correctly classified, figures much better than those obtained with NSE or S100B, in the same study's population. H-FABP appears to be a valid serum biomarker for the early diagnosis of stroke. Further studies on large cohorts of patients are warranted.     

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.     

The Expression and Significance of Neuronal Iconic Proteins in Podocytes

Growing evidence suggests that there are many common cell biological features shared by neurons and podocytes; however, the mechanism of podocyte foot process formation remains unclear. Comparing the mechanisms of process formation between two cell types should provide useful guidance from the progress of neuron research. Studies have shown that some mature proteins of podocytes, such as podocin, nephrin, and synaptopodin, were also expressed in neurons. In this study, using cell biological experiments and immunohistochemical techniques, we showed that some neuronal iconic molecules, such as Neuron-specific enolase, nestin and Neuron-specific nuclear protein, were also expressed in podocytes. We further inhibited the expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 by Small interfering RNA in cultured mouse podocytes and observed the significant morphological changes in treated podocytes. When podocytes were treated with Adriamycin, the protein expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 decreased over time. Meanwhile, the morphological changes in the podocytes were consistent with results of the Small interfering RNA treatment of these proteins. The data demonstrated that neuronal iconic proteins play important roles in maintaining and regulating the formation and function of podocyte processes.     

La protéine S100B, marqueur biologique des lésions cérébrales aiguës

S100B protein is a constitutive protein of glial cells, whose physiological functions are both intracellular, i.e. intracytosolic calcium binding, and extracellular, e.g. by promoting neuritic proliferation and/or neuronal apoptosis. Due to specificity of its cellular expression, S100B protein is a useful biological marker of neurological disorders, such as ischemic or haemorragic stroke, and traumatic head injury. This brief review the contribution of S100B measurement in biological fluids (cerebrospinal fluid, blood) to diagnosis, follow-up, and prognosis of acute brain damage events.     

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

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

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     

Increased Nervous System-Specific Enolases in Rat Plasma and Cerebrospinal Fluid in Bilirubin Encephalopathy Detected by an Enzyme Immunoassay

Abstract: Three forms of enolase isozymes (αα, αγ, and γγ), including nervous system-specific forms, were measured in the cerebrospinal fluid and the blood plasma of jaundiced or nonjaundiced infant rats by means of enzyme immunoassay systems capable of detecting each form of enolase at the 1 amol (10⁻¹⁸ mol) level. Average enolase levels in cerebrospinal fluid in normal rat were 2.0, 0.2 and 0.1 pmol/ml for αα, αγ, and γγ forms, respectively. Levels of αγ and γγ forms (nervous system-specific enolases; NSE) in jaundiced rats, which suffer Purkinje cell degeneration due to the inborn hyperbilirubinemia, were three to four times as high as the normal values. When kernicterus was induced in jaundiced rats by an injection of bucolome, the NSE level in cerebrospinal fluid was elevated up to more than 30-fold the control, together with a significantly higher level of αγ form in blood plasma. These results suggest that assays of NSE in the cerebrospinal fluid or the blood plasma are helpful in detecting neuronal damage in the central nervous system.     

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

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 para-benzoquinone 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.     

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.     

Recovering circulating extracellular or cell-free RNA from bodily fluids

The presence of extracellular circulating or cell-free RNA in biological fluids is becoming a promising diagnostic tool for non invasive and cost effective cancer detection. Extracellular RNA or miRNA as biological marker could be used either for the early detection and diagnosis of the disease or as a marker of recurrence patterns and surveillance. In this review article, we refer to the origin of the circulating extracellular RNA, we summarise the data on the biological fluids (serum/plasma, saliva, urine, cerebrospinal fluid and bronchial lavage fluid) of patients suffering from various types of malignancies reported to contain a substantial amount of circulating extracellular (or cell-free) RNAs and we discuss the appropriate reagents and methodologies needed to be employed in order to obtain RNA material of high quality and integrity for the majority of the experimental methods used in RNA expression analysis. Furthermore, we discuss the advantages and disadvantages of the RT-PCR or microarray methodology which are the methods more often employed in procedures of extracellular RNA analysis.     

Brain-derived proteins in the CSF, do they correlate with brain pathology in CJD?

Background

Brain derived proteins such as 14-3-3, neuron-specific enolase (NSE), S 100b, tau, phosphorylated tau and Aβ_1–42 were found to be altered in the cerebrospinal fluid (CSF) in Creutzfeldt-Jakob disease (CJD) patients. The pathogenic mechanisms leading to these abnormalities are not known, but a relation to rapid neuronal damage is assumed. No systematic analysis on brain-derived proteins in the CSF and neuropathological lesion profiles has been performed.

Methods

CSF protein levels of brain-derived proteins and the degree of spongiform changes, neuronal loss and gliosis in various brain areas were analyzed in 57 CJD patients.

Results

We observed three different patterns of CSF alteration associated with the degree of cortical and subcortical changes. NSE levels increased with lesion severity of subcortical areas. Tau and 14-3-3 levels increased with minor pathological changes, a negative correlation was observed with severity of cortical lesions. Levels of the physiological form of the prion protein (PrP^c) and Aβ_1–42 levels correlated negatively with cortical pathology, most clearly with temporal and occipital lesions.

Conclusion

Our results indicate that the alteration of levels of brain-derived proteins in the CSF does not only reflect the degree of neuronal damage, but it is also modified by the localization on the brain pathology. Brain specific lesion patterns have to be considered when analyzing CSF neuronal proteins.     

Peptidylarginine deiminase modulates the physiological roles of enolase via citrullination: links between altered multifunction of enolase and neurodegenerative diseases

The citrullination of enolase by PAD (peptidylarginine deiminase) has emerged as an important post-translational modification in human disorders; however, the physiological function of citrullination remains unknown. In the present study, we report that citrullination diversely regulates the biological functions of ENO1 (α-enolase) and NSE (neuron-specific enolase). We developed three mouse IgG1 monoclonal antibodies with specificity to the following: (i) citrullination of Arg9 of ENO1 [ENO1Cit9; anti-CE1 (citrullinated enolase 1) antibody]; (ii) citrullination of Arg9 in ENO1 and NSE (ENO1Cit9/NSECit9; anti-CE1/2 antibody); and (iii) citrullination of Arg429 of NSE (NSECit429; anti-CE2 antibody). Regardless of the total protein expression level, the levels of ENO1Cit9 and NSECit429 were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in the frontal cortex of patients with sporadic Creutzfeldt-Jakob disease and Alzheimer's disease compared with controls. In a time- and dose-dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by Ca2+-dependent calpain-1, and leupeptin (calpain inhibitor I) abrogated this degradation. Surprisingly, using an affinity assay, the citrullination of enolase enhanced its plasminogen-binding affinity, which was blocked by the lysine analogue ?-aminocaproic acid. These findings suggest that PAD-mediated citrullination regulates the diverse physiological activities of enolase and that CE may be a candidate diagnostic/prognostic factor for degenerative diseases.     

Co-localization of neuron-specific enolase-like and kallikrein-like immunoreactivity in ductal and tubular epithelium of sheep salivary gland and kidney

Neuron-specific enolase-like and kallikrein-like immunoreactivity was found to be co-localized in the ductal elements of the submandibular gland and in the more distal portions of the nephron in the kidney of newborn lambs. Some glomerular peripolar cells in the kidney were immunopositive for neuron-specific enolase without detectable kallikrein-like immunoreactivity.     

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.     

Immunocytochemical studies on parafollicular cells of various mammals

Using specific antisera, calcitonin, calcitonin gene-related peptide (CGRP), somatostatin as well as neuron-specific enolase, chromogranin, secretory peptide I and calbindin (vitamin D-dependent calcium-binding protein) were looked for in parafollicular cells of rats, Syrian hamsters, Mongolian gerbils, mice, guinea pigs, rabbits and pigs. Calcitonin and CGRP were most invariably present in various species. Somatostatin was absent in mice and Mongolian gerbils and present in variable amounts in the remaining species. Neuron-specific enolase could not be detected in rabbits, while in the pigs and the Mongolian gerbils it could be demonstrated only in some parafollicular cells. Calbindin was present exclusively in parafollicular cells of guinea pigs. Chromogranin and secretory protein-I were present only in some animal species.     

Detection of 14-3-3zeta in cerebrospinal fluid following experimentally evoked seizures

Surrogate and peripheral (bio)markers of neuronal injury may be of value in assessing effects of seizures on the brain or epilepsy development following trauma. The presence of 14-3-3 isoforms in cerebrospinal fluid (CSF) is a diagnostic indicator of Creutzfeldt-Jakob disease but these proteins may also be present following acute neurological insults. Here, we examined neuronal and 14-3-3 proteins in CSF from rats after seizures. Seizures induced by intra-amygdala microinjection of 0.1 microg kainic acid (KA) caused damage which was mainly restricted to the ipsilateral CA3 subfield of the hippocampus. 14-3-3zeta was detected at significant levels in CSF sampled 4 h after seizures compared with near absence in control CSF. Neuron-specific nuclear protein (NeuN) was also elevated in CSF in seizure rats. CSF 14-3-3zeta levels were significantly lower in rats treated with 0.01 microg KA. These data suggest the presence of 14-3-3zeta within CSF may be a biomarker of acute seizure damage.     

Immunoaffinity liquid chromatography-tandem mass spectrometry detection of nitrotyrosine in biological fluids: development of a clinically translatable biomarker

Measurement of nitrotyrosine levels in biological fluids can serve as a biomarker for oxidative/nitrative damage arising from formation of reactive nitrogen species, including peroxynitrite. Peroxynitrite is formed by the reaction of the superoxide radical (O2.-) with the nitric oxide radical (.NO) that is generated by nitric oxide synthase (NOS). This article describes an immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 3-nitrotyrosine at very low (picomolar) levels. Incorporation of a pronase digestion step prior to the immunoaffinity LC-MS/MS allowed for measuring not only free amino acid but also protein 3-nitrotyrosine in biological fluids. The use of an in-line antibody column allowed for increased specificity as compared with previously reported assays. The assay is linear over a range of 5 to 500 pg/ml (0.022-2.20 nM, r(2)=0.9987), with the lower detection limit being 5 pg/ml. In addition to its increased sensitivity and specificity, this assay showed great nitrotyrosine recovery from biological fluids when either nitrotyrosine or nitrotyrosine-containing peptides were added exogenously. The utility of this assay for nitrotyrosine as a clinically translatable biomarker was demonstrated by quantifying both free and total nitrotyrosine levels in various biological fluids, including urine, plasma, serum, cerebrospinal fluid (CSF), and synovial fluid (SF) from both preclinical species and human subjects. Thus, whether in an animal model of human disease or in a clinical setting, the quantification of nitrotyrosine levels should provide support for NOS-driven pathology and its blockade following therapeutic intervention.     

Detection of 14-3-3ζ in cerebrospinal fluid following experimentally evoked seizures

Surrogate and peripheral (bio)markers of neuronal injury may be of value in assessing effects of seizures on the brain or epilepsy development following trauma. The presence of 14-3-3 isoforms in cerebrospinal fluid (CSF) is a diagnostic indicator of Creutzfeldt–Jakob disease but these proteins may also be present following acute neurological insults. Here, we examined neuronal and 14-3-3 proteins in CSF from rats after seizures. Seizures induced by intra-amygdala microinjection of 0.1 µg kainic acid (KA) caused damage which was mainly restricted to the ipsilateral CA3 subfield of the hippocampus. 14-3-3ζ was detected at significant levels in CSF sampled 4 h after seizures compared with near absence in control CSF. Neuron-specific nuclear protein (NeuN) was also elevated in CSF in seizure rats. CSF 14-3-3ζ levels were significantly lower in rats treated with 0.01 µg KA. These data suggest the presence of 14-3-3ζ within CSF may be a biomarker of acute seizure damage.