Aspartoacylase-lacZ knockin mice: an engineered model of Canavan disease

Canavan Disease (CD) is a recessive leukodystrophy caused by loss of function mutations in the gene encoding aspartoacylase (ASPA), an oligodendrocyte-enriched enzyme that hydrolyses N-acetylaspartate (NAA) to acetate and aspartate. The neurological phenotypes of different rodent models of CD vary considerably. Here we report on a novel targeted aspa mouse mutant expressing the bacterial β-Galactosidase (lacZ) gene under the control of the aspa regulatory elements. X-Gal staining in known ASPA expression domains confirms the integrity of the modified locus in heterozygous aspa lacZ-knockin (aspa(lacZ/+)) mice. In addition, abundant ASPA expression was detected in Schwann cells. Homozygous (aspa(lacZ/lacZ)) mutants are ASPA-deficient, show CD-like histopathology and moderate neurological impairment with behavioural deficits that are more pronounced in aspa(lacZ/lacZ) males than females. Non-invasive ultrahigh field proton magnetic resonance spectroscopy revealed increased levels of NAA, myo-inositol and taurine in the aspa(lacZ/lacZ) brain. Spongy degeneration was prominent in hippocampus, thalamus, brain stem, and cerebellum, whereas white matter of optic nerve and corpus callosum was spared. Intracellular vacuolisation in astrocytes coincides with axonal swellings in cerebellum and brain stem of aspa(lacZ/lacZ) mutants indicating that astroglia may act as an osmolyte buffer in the aspa-deficient CNS. In summary, the aspa(lacZ) mouse is an accurate model of CD and an important tool to identify novel aspects of its complex pathology.     

Are Astrocytes the Missing Link Between Lack of Brain Aspartoacylase Activity and the Spongiform Leukodystrophy in Canavan Disease?

Canavan disease (CD) is a genetic degenerative brain disorder associated with mutations of the gene encoding aspartoacylase (ASPA). In humans, the CD syndrome is marked by early onset, hydrocephalus, macroencephaly, psychomotor retardation, and spongiform myelin sheath vacuolization with progressive leukodystrophy. Metabolic hallmarks of the disease include elevated N-acetylaspartate (NAA) levels in brain, plasma and CSF, along with daily excretion of large amounts of NAA and its anabolic metabolite, N-acetylaspartylglutamate (NAAG). Of the observed neuropathies, the most important appears to be the extensive demyelination that interferes with normal neuronal signaling. However, finding the links between the lacks of ASPA activity in oligodendrocytes, the buildup of NAA in white matter (WM) and the mechanisms underlying the edematous spongiform leukodystrophy have remained elusive. In this analytical review we consider what those links might be and propose that in CD, the pathological buildup of NAA in limited WM extracellular fluid (ECF) is responsible for increased ECF osmotic–hydrostatic pressure and initiation of the demyelination process. We also hypothesize that NAA is not directly liberated by neurons in WM as it is in gray matter, and that its source in WM ECF is solely as a product of the catabolism of axon-released NAAG at nodes of Ranvier by astrocyte NAAG peptidase after it has docked with the astrocyte surface metabotropic glutamate receptor 3. This hypothesis ascribes for the first time a possible key role played by astrocytes in CD, linking the lack of ASPA activity in myelinating oligodendrocytes, the pathological buildup of NAA in WM ECF, and the spongiform demyelination process. It also offers new perspectives on the cause of the leukodystrophy in CD, and on possible treatment strategies for this inherited metabolic disease. CD, a rare genetic disorder that compromises a physiologically important tri-cellular brain metabolic system.     

A mutation of aspartoacylase gene in a Turkish patient with Canavan disease

Canavan disease (CD) is an autosomal recessive inherited disorder characterized by spongy degeneration of the brain. The deficiency of aspartoacylase (ASPA), resulting in the accumulation of N-acetyl aspartic acid (NAA) in the brain, plays an important role in the pathogenesis of the disease. The cardinal features of this neurodegenerative disease are macrocephaly, mental retardation, and hypotonia. Magnetic resonance imaging (MRI) of the brain generally shows diffuse white matter degeneration and also elevated excretion of urinary NAA is usually seen. A large number of mutations were identified to date. We report here a 9 months old girl with Canavan Disease and a homozygous c.79G>A mutation in the ASPA gene, detected for the first time in Turkish population.     

Decreased NAA in Gray Matter is Correlated with Decreased Availability of Acetate in White Matter in Postmortem Multiple Sclerosis Cortex

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS) which leads to progressive neurological disability. Our previous studies have demonstrated mitochondrial involvement in MS cortical pathology and others have documented decreased levels of the neuronal mitochondrial metabolite N-acetyl aspartate (NAA) in the MS brain. While NAA is synthesized in neurons, it is broken down in oligodendrocytes into aspartate and acetate. The resulting acetate is incorporated into myelin lipids, linking neuronal mitochondrial function to oligodendrocyte-mediated elaboration of myelin lipids in the CNS. In the present study we show that treating human SH-SY5Y neuroblastoma cells with the electron transport chain inhibitor antimycin A decreased levels of NAA as measured by HPLC. To better understand the significance of the relationship between mitochondrial function and levels of NAA and its breakdown product acetate on MS pathology we then quantitated the levels of NAA and acetate in MS and control postmortem tissue blocks. Regardless of lesion status, we observed that levels of NAA were decreased 25 and 32 % in gray matter from parietal and motor cortex in MS, respectively, compared to controls. Acetate levels in adjacent white matter mirrored these decreases as evidenced by the 36 and 45 % reduction in acetate obtained from parietal and motor cortices. These data suggest a novel mechanism whereby mitochondrial dysfunction and reduced NAA levels in neurons may result in compromised myelination by oligodendrocytes due to decreased availability of acetate necessary for the synthesis of myelin lipids.     

Cytotoxic and complement-binding activity of rabbit antisera against the cortex and cerebral white matter of rats and humans]

The cytotoxicity and complement-fixation activity of rabbit antisera against rat and human brain cortex and white matter was tested against mouse and rat thymocytes and bone marrow cells. The cytotoxic test proved to be more sensitive and accurate. The cytotoxins to rodent thymocytes were found in the antisera against human brain cortex only. At the same time cytotoxic antibodies were revealed both in the antisera rat brain cortex and white matter; but the former contained much more cytotoxic antibodies than the letter. After absorption with the same antigen the antisera against rat brain cortex lost their cytotoxic effect, but retained it in case of absorption with the white matter.     

N-acetylaspartate in the vertebrate brain: metabolism and function

N-Acetyl-l-aspartate (NAA) is an amino acid that is present in the vertebrate brain. Its concentration is one of the highest of all free amino acids and, although NAA is synthesized and stored primarily in neurons, it cannot be hydrolyzed in these cells. Furthermore, neuronal NAA is dynamic and turns over more than once each day by virtue of its continuous efflux, in a regulated intercompartmental cycling via extracellular fluids, between neurons and a second compartment in oligodendrocytes. The metabolism of NAA, between its anabolic compartment in neurons and its catabolic compartment in oligodendrocytes, and its possible physiological role in the brain has been the subject of much speculation. There are two human inborn errors in metabolism of NAA. One is Canavan disease (CD), in which there is a buildup of NAA (hyperacetylaspartia) and associated spongiform leukodystrophy, caused by a lack of aspartoacylase activity. The other is a singular human case of lack of NAA (hypoacetylaspartia), where the enzyme that synthesizes NAA is apparently absent. There are two animal models currently available for studies of CD. One is a rat with a natural deletion of the catabolic enzyme, and the other a gene knockout mouse. In addition to the presence of NAA in neurons, its prominence in ¹H nuclear magnetic resonance spectroscopic studies has led to its wide use in diagnostic human medicine as both an indicator of brain pathology and of disease progression in a variety of CNS diseases. In this review, various hypotheses regarding the metabolism of NAA and its possible role in the CNS are evaluated. Based on this analysis, it is concluded that although NAA may have several functions in the CNS, an important role of the NAA intercompartmental system is osmoregulatory, and in this role it may be the primary mechanism for the removal of intracellular water, against a water gradient, from myelinated neurons.     

Developmental and regional distribution of aspartoacylase in rat brain tissue

The function of N-acetyl-aspartate (NAA), a predominant molecule in the brain, has not yet been determined. However, NAA is commonly used as a putative marker of viable neurones. To investigate the possible function of NAA, we determined the anatomical, developmental and cellular distribution of aspartoacylase, which catalyses the hydrolysis of NAA. Levels of aspartoacylase activity were measured during postnatal development in several brain regions. The differential distribution of aspartoacylase activity in purified populations of cells derived from the rat CNS was also investigated. The developmental and anatomical distribution of aspartoacylase correlated with the maturation of white matter tracts in the rat brain. Activity increased markedly after 7 days and coincided with the time course for the onset of myelination in the rat brain. Gray matter showed little activity or developmental trend. There was a 60-fold excess in optic nerve (a white matter tract) when compared with cortex at 21 days of development. In the adult brain there was a 18-fold difference in corpus callosum compared with cortex (stripped of corpus callosum). Cellular studies demonstrated that purified cortical neurons and cerebellar granular neurones have no activity. Primary O-2A progenitor cells had moderate activity, with three-fold higher activity in immature oligodendrocyte and 13-fold increase in mature oligodendrocytes (myelinating cells of the CNS). The highest activity was seen in type-2 astrocytes (20-fold difference compared with O-2A progenitors) derived from the same source. Aspartoacylase activity increased with time in freshly isolated astrocytes, with significantly higher activity after 15 days in culture. We conclude that aspartoacylase activity in the developing postnatal brain corresponds with maturation of myelination, and that the cellular distribution is limited to glial cells.     

A modelling approach to explore some hypotheses of the failure of neuroprotective trials in ischemic stroke patients

Ischemic stroke is the third cause of death in industrialised countries, but no satisfactory treatment is currently available. The hundreds of neuroprotective drugs developed to block the ischemic cascade gave very promising results in animal models but the clinical trials performed with these drugs showed no beneficial effects in stroke patients. Many hypotheses were advanced to explain this discrepancy, among which the morphological and functional differences between human and rodent brains. This discrepancy could be partly due to the differences in white matter and glial cell proportions between human and rodent brains. In order to test this hypothesis, we built a mathematical model of the main early pathophysiological mechanisms of stroke in rodent and in human brains. This model is a two-scale model and relies on a set of ordinary differential equations. We built two versions of this model (for human and rodent brains) differing in their white matter and glial cell proportions. Then, we carried out in silico experiments with various neuroprotective drugs. The simulation results obtained with a sodium channel blocker show that the proportion of penumbra recovery is much higher in rodent than in human brain and the results are similar with some other neuroprotective drugs tested during phase III trials. This in silico investigation suggests that the proportions of glial cells and white matter have an influence on neuroprotective drug efficacy. It reinforces the hypothesis that histological and morphological differences between rodent and human brains can partly explain the failure of these agents in clinical trials.     

The subcellular localization of the carbohydrate epitope 3-fucosyl-N-acetyllactosamine is different in normal and reactive astrocytes.

The carbohydrate epitope 3-fucosyl-N-acetyllactosamine (CD15) is involved in cell-to-cell recognition processes in various tissues. In the present study the subcellular localization of CD15 was immunocytochemically studied in normal and pathological central nervous system fiber tracts of humans and rats. In normal human white matter of the brain, CD15 immunoreactivity was found on the cell surface of astrocytes and within the cytoplasm of oligodendrocytes. In freshly demyelinated lesions of two human diseases (central pontine myelinolysis and multiple sclerosis) strong cytoplasmic CD15 staining was observed in reactive astrocytes. In normal rats CD15 immunostaining was restricted to the surface of astrocytes. In crush-induced lesions of rat optic nerves, however, astrocytes showed a cytoplasmic localization of CD15, 4 and 6 days after injury. In conclusion, abnormal localization of CD15 in reactive astrocytes may be related to altered functional states of these cells during disease processes.     

Lessons from genetically engineered animal models XI. Novel mouse models to study pathogenic mechanisms of Crohn's disease

Crohn's Disease (CD) affects more than 500,000 individuals in the United States and represents the second most common chronic inflammatory disorder after rheumatoid arthritis. Although major advances have been made in defining the basic mechanisms underlying chronic intestinal inflammation, the precise etiopathogenesis of CD remains unknown. We have recently characterized two novel mouse models of enteritis that express a CD-like phenotype, namely the TNF DeltaARE model of tumor necrosis factor (TNF) overexpression and the SAMP1/Yit model of spontaneous ileitis. The unique feature of these models is that they closely resemble CD for location and histopathology. These genetically manipulated new models of intestinal inflammation offer a powerful tool to investigate potential causes of human disease and may allow the development of novel disease-modifying therapeutic modalities for the treatment of CD.     

Effect of antibodies to rat and human cerebral cortex and white matter on heterologous T- and B-cells]

A study was made of the cytotoxic and complement-fixation activity of the antisera to the cortex and white matter of the rat and human brain upon the mouse and rat thymus and bone marrow cells. The cytotoxicity test proved to be more sensitive and precise. Cytotoxins to rodent thymocytes were revealed on ly in the antisera against the human brain cortex; at the same time they were revealed both in the antisera against the cortex and against the white matter of the rat brain (much more was found in the former). The sera against the rat brain cortex lost their cytotoxicity after the exhaustion with the same antigen, but retained it when the exhaustion was carried out by the white matter.     

Developmental regulation of group I metabotropic glutamate receptors in the premature brain and their protective role in a rodent model of periventricular leukomalacia

Cerebral white matter injury in premature infants, known as periventricular leukomalacia (PVL), is common after hypoxia-ischemia (HI). While ionotropic glutamate receptors (iGluRs) can mediate immature white matter injury, we have previously shown that excitotoxic injury to premyelinating oligodendrocytes (preOLs) in vitro can be attenuated by group I metabotropic glutamate receptor (mGluR) agonists. Thus, we evaluated mGluR expression in developing white matter in rat and human brain, and tested the protective efficacy of a central nervous system (CNS)-penetrating mGluR agonist on injury to developing oligodendrocytes (OLs) in vivo. Group I mGluRs (mGluR1 and mGluR5) were strongly expressed on OLs in neonatal rodent cerebral white matter throughout normal development, with highest expression early in development on preOLs. Specifically at P6, mGluR1 and mGLuR5 were most highly expressed on GalC-positive OLs compared to neurons, axons, astrocytes and microglia. Systemic administration of (1S,3R) 1-aminocyclopentane-trans-1,3,-dicarboxylic acid (ACPD) significantly attenuated the loss of myelin basic protein in the white matter following HI in P6 rats. Assessment of postmortem human tissue showed both mGluR1 and mGluR5 localized on immature OLs in white matter throughout development, with mGluR5 highest in the preterm period. These data indicate group I mGluRs are highly expressed on OLs during the peak period of vulnerability to HI and modulation of mGluRs is protective in a rodent model of PVL. Group I mGluRs may represent important therapeutic targets for protection from HI-mediated white matter injury.     

An In Vivo Kinetic Model with L-[35S]Methionine for the Determination of Local Cerebral Rates for Methionine Incorporation into Protein in the Rat

A method has been developed for the simultaneous in vivo measurement of local rates for methionine incorporation into cerebral protein in the rat. It is based on the use of L-[35S]methionine as a tracer for reflecting the bidirectional exchange of methionine between plasma and brain and its incorporation into cerebral protein, using a dynamic three-compartment model. An operational equation based on this model has been derived in terms of determinable variables. The method has been applied to the normal freely moving rat and to the rat under chloral hydrate anesthesia. In the freely moving rat, the values of methionine incorporation into cerebral protein in the gray matter vary widely from structure to structure (50-300 nmol/100 g/min), with the highest values in structures related to neurosecretory functions, e.g., supraoptic and paraventricular nuclei. The values for white matter are more uniform (24-28 nmol/100 g/min) at levels approximately six- to seven-fold lower than for gray matter. Chloral hydrate anesthesia depresses the rate of methionine incorporation in all the structures examined. Anesthesia did not reduce the heterogeneity normally present within gray matter.     

A quantitative model for differential motility of gliomas in grey and white matter

We have extended a mathematical model of gliomas based on proliferation and diffusion rates to incorporate the effects of augmented cell motility in white matter as compared to grey matter. Using a detailed mapping of the white and grey matter in the brain developed for a MRI simulator, we have been able to simulate model tumours on an anatomically accurate brain domain. Our simulations show good agreement with clinically observed tumour geometries and suggest paths of submicroscopic tumour invasion not detectable on CT or MRI images. We expect this model to give insight into microscopic and submicroscopic invasion of the human brain by glioma cells. This method gives insight in microscopic and submicroscopic invasion of the human brain by glioma cells. Additionally, the model can be useful in defining expected pathways of invasion by glioma cells and thereby identify regions of the brain on which to focus treatments.     

Regional Variation in the Levels of Transferrin in the CNS of Normal and Myelin-Deficient Rats

Transferrin (Tf), the iron mobilization protein, is synthesized mainly in the liver. Recently, both Tf and a mRNA for Tf have been demonstrated in oligodendrocytes in the rat brain. The present study used a biochemical assay for determining the levels of Tf in various brain regions of normal rats compared with the level of those obtained from rats with a genetic mutation characterized by an almost complete failure to develop myelin. In myelin-deficient (md) rats, no Tf-positive oligodendrocytes were seen immunohistochemically in the gray or white matter of the CNS. Quantitatively, levels of Tf throughout the CNS of the md rat were decreased to approximately 5% of the normal values despite a normal hepatic synthetic rate. In the normal rat brain, the cerebellum contained the highest concentration of Tf, followed by the pons, the cerebral cortex, and the caudate-putamen, with the latter two sites being similar. Regional variation in the amount of Tf was in general agreement with published reports on the variation of iron and Tf receptor levels in the CNS. Immunohistochemical examination with antiserum to galactocerebroside (a myelin-specific lipid) was used for extending biochemical reports that glycolipid-synthesizing enzymes are deficient in md rats. No immunostaining in the md rat was observed following immunoreaction for galactocerebroside, whereas white matter oligodendrocytes were intensely marked in the normal rat. Robust astrogliosis was present in both the gray and white matter of the md rats. It is not known at present whether the ability to accumulate Tf is necessary for oligodendrocytic survival or if Tf accumulation is more directly related to myelinogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA Editing of the Glutamate Receptor Subunits GluR2 and GluR6 in Human Brain Tissue

Abstract: Editing of mRNA in the coding region of the second transmembrane domain of glutamate receptor subunits GluR2, GluR5, and GluR6 involves a change of the base A in genomic DNA to the base G in mRNA as described in rat brain. To determine whether this reaction occurs in humans as well as rats, we studied RNA editing of GluR2 and GluR6 in human brain. We compared the extent of editing in controls and cases with Huntington's disease. To assay the extent of editing in brain RNA, first strand cDNA was amplified using the polymerase chain reaction yielding a product across the region of the second transmembrane spanning segment in which editing takes place in rats. The PCR product was incubated with the restriction enzyme BbvI, which recognizes the sequence GCAGC present in the nonedited sequence of the mRNA in subunits GluR2 and GluR6. Thus, BbvI cuts the nonedited version but leaves the edited version intact. As in the rat, the GluR2 subunit mRNA was completely edited in human brain. The GluR6 subunit was nearly completely edited in all gray matter structures investigated including cortex, striatum, thalamus, hippocampus, amygdala, and cerebellum with extent of editing ranging from 89% in the cerebellum to 95% in the cortex and striatum. No significant differences in the extent of RNA editing were apparent in control versus Huntington's disease brains. To compare the extent of editing in neurons and glia in the brain, editing in cerebral cortex (predominantly gray matter and thus neurons) was compared with editing in corpus callosum (white matter and thus nearly completely glial cells). In white matter, GluR2 was completely edited, whereas GluR6 was only ～10% edited compared with ～90% edited in gray matter. Thus, these studies indicate that RNA editing is seen in human brain as well as rat brain and that the extent of editing is similar in Huntington's disease compared with controls. The differences in editing in white matter for GluR6, but not for GluR2, suggest that different templates could be subject to different editing activities that undergo tissue-specific regulation.     

Accumulation of N-acetyl-L-aspartate in the brain of the tremor rat, a mutant exhibiting absence-like seizure and spongiform degeneration in the central nervous system

The tremor rat is a mutant that exhibits absence-like seizure and spongiform degeneration in the CNS. By positional cloning, a genomic deletion was found within the critical region in which the aspartoacylase gene is located. Accordingly, no aspartoacylase expression was detected in any of the tissues examined, and abnormal accumulation of N-acetyl-L-aspartate (NAA) was shown in the mutant brain, in correlation with the severity of the vacuole formation. Therefore, the tremor rat may be regarded as a suitable animal model of human Canavan disease, characterized by spongy leukodystrophy that is caused by aspartoacylase deficiency. Interestingly, direct injection of NAA into normal rat cerebroventricle induced 4- to 10-Hz polyspikes or spikewave-like complexes in cortical and hippocampal EEG, concomitantly with behavior characterized by sudden immobility and staring. These results suggested that accumulated NAA in the CNS would induce neuroexcitation and neurodegeneration directly or indirectly.     

Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase

Despite its growing use as a radiological indicator of neuronal viability, the biological function of N-acetylaspartate (NAA) has remained elusive. This is due in part to its unusual metabolic compartmentalization wherein the synthetic enzyme occurs in neuronal mitochondria whereas the principal metabolizing enzyme, N-acetyl-L-aspartate amidohydrolase (aspartoacylase), is located primarily in white matter elements. This study demonstrates that within white matter, aspartoacylase is an integral component of the myelin sheath where it is ideally situated to produce acetyl groups for synthesis of myelin lipids. That it functions in this manner is suggested by the fact that myelin lipids of the rat optic system are well labeled following intraocular injection of [14C-acetyl]NAA. This is attributed to uptake of radiolabeled NAA by retinal ganglion cells followed by axonal transport and transaxonal transfer of NAA into myelin, a membrane previously shown to contain many lipid synthesizing enzymes. This study identifies a group of myelin lipids that are so labeled by neuronal [14C]NAA, and demonstrates a different labeling pattern from that produced by neuronal [14C]acetate. High performance liquid chromatographic analysis of the deproteinated soluble materials from the optic system following intraocular injection of [14C]NAA revealed only the latter substance and no radiolabeled acetate, suggesting little or no hydrolysis of NAA within mature neurons of the optic system. These results suggest a rationale for the unusual compartmentalization of NAA metabolism and point to NAA as a neuronal constituent that is essential for the formation and/or maintenance of myelin. The relevance of these findings to Canavan disease is discussed.     

Focus on RNA isolation: obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue

MicroRNAs (miRNAs) are present in all known plant and animal tissues and appear to be somewhat concentrated in the mammalian nervous system. Many different miRNA expression profiling platforms have been described. However, relatively little research has been published to establish the importance of 'upstream' variables in RNA isolation for neural miRNA expression profiling. We tested whether apparent changes in miRNA expression profiles may be associated with tissue processing, RNA isolation techniques, or different cell types in the sample. RNA isolation was performed on a single brain sample using eight different RNA isolation methods, and results were correlated using a conventional miRNA microarray and then cross-referenced to Northern blots. Differing results were seen between samples obtained using different RNA isolation techniques and between microarray and Northern blot results. Another complication of miRNA microarrays is tissue-level heterogeneity of cellular composition. To investigate this phenomenon, miRNA expression profiles were determined and compared between highly-purified primary cerebral cortical cell preparations of rat primary E15-E18 neurons versus rat primary E15-E18 astrocytes. Finally, to assess the importance of dissecting human brain gray matter from subjacent white matter in cerebral cortical studies, miRNA expression profiles were compared between gray matter and immediately contiguous white matter. The results suggest that for microarray studies, cellular composition is important, and dissecting white matter from gray matter improves the specificity of the results. Based on these data, recommendations for miRNA expression profiling in neural tissues, and considerations worthy of further study, are discussed.     

MEASUREMENT OF NEURON-SPECIFIC (NSE) AND NON-NEURONAL (NNE) ISOENZYMES OF ENOLASE IN RAT, MONKEY AND HUMAN NERVOUS TISSUE

Four double antibody solid-phase radioimmunoassay systems are described for the measurement of neuron-specific enolase (NSE) and non-neuronal enolase (NNE) from rat, monkey and human brain tissue. NSE and NNE are antigenically distinct, making their respective assays specific. The levels of neuronal and non-neuronal enolase (an enolase recently shown to be localized in glial cells) are determined in various regions of rat, monkey and human nervous system. Both neuronal and glial enolases are major proteins of brain tissue with each representing about 1.5% of total brain soluble protein. NSE levels are highest and NNE levels lowest in brain areas having a high proportion of grey matter, such as the cerebral cortex. The reverse is true for areas high in white matter, such as the pyramidal tract and the corpus callosum. Peripheral nervous system levels of NSE are much lower than those of brain with the spinal cord intermediate between the two. Radioimmunological and immunocytochemical data show that neuron-specific enolase is also present in neuroendocrine cells located in non-nervous tissue, which include pinealocytes, parafollicular cells of the thyroid, adrenal medullary chromaffin cells, glandular cells of the pituitary and Islet of Langerhans cells in the pancreas. Unlike neurons, these cells also contain non-neuronal enolase in high amounts.