Asynchrony in Peritumoral Resting-State Blood Oxygen Level-Dependent fMRI Predicts Meningioma Grade and Invasion.

BACKGROUND AND PURPOSE: Meningioma grade is determined by histologic analysis, with detectable brain invasion resulting in a diagnosis of grade II or III tumor. However, tissue undersampling is a common problem, and invasive parts of the tumor can be missed, resulting in the incorrect assignment of a lower grade. Radiographic biomarkers may be able to improve the diagnosis of grade and identify targets for biopsy. Prior work in patients with gliomas has shown that the resting-state blood oxygen level-dependent fMRI signal within these tumors is not synchronous with normal brain. We hypothesized that blood oxygen level-dependent asynchrony, a functional marker of vascular dysregulation, could predict meningioma grade. MATERIALS AND METHODS: We identified 25 patients with grade I and 11 patients with grade II or III meningiomas. Blood oxygen level-dependent time-series were extracted from the tumor and the radiographically normal control hemisphere and were included as predictors in a multiple linear regression to generate a blood oxygen level-dependent asynchrony map, in which negative values signify synchronous and positive values signify asynchronous activity relative to healthy brain. Masks of blood oxygen level-dependent asynchrony were created for each patient, and the fraction of the mask that extended beyond the contrast-enhancing tumor was computed. RESULTS: The spatial extent of blood oxygen level-dependent asynchrony was greater in high (grades II and III) than in low (I) grade tumors (P < 0.001) and could discriminate grade with high accuracy (area under the curve = 0.88). CONCLUSIONS: Blood oxygen level-dependent asynchrony radiographically discriminates meningioma grade and may provide targets for biopsy collection to aid in histologic diagnosis.

Roles of HAUSP-mediated p53 regulation in central nervous system development.

The deubiquitinase HAUSP (herpesvirus-associated ubiquitin-specific protease; also called USP7) has a critical role in regulating the p53-Mdm2 (murine double minute 2) pathway. By using the conventional knockout approach, we previously showed that hausp inactivation leads to early embryonic lethality. To fully understand the physiological functions of hausp, we have generated mice lacking hausp specifically in the brain and examined the impacts of this manipulation on brain development. We found that deletion of hausp in neural cells resulted in neonatal lethality. The brains from these mice displayed hypoplasia and deficiencies in development, which were mainly caused by p53-mediated apoptosis. Detailed analysis also showed an increase of both p53 levels and p53-dependent transcriptional activation in hausp knockout brains. Notably, neural cell survival and brain development of hausp-mutant mice can largely be restored in the p53-null background. Nevertheless, in contrast to the case of mdm2- and mdm4 (murine double minute 4)-mutant mice, inactivation of p53 failed to completely rescue the neonatal lethality of these hausp-mutant mice. These results indicate that HAUSP-mediated p53 regulation is crucial for brain development, and also suggest that both the p53-dependent and the p53-independent functions of HAUSP contribute to the neonatal lethality of hausp-mutant mice.

Selective destruction of glioblastoma cells by interference with the activity or expression of ATF5.

Glioblastoma multifome is the most common and most aggressive primary brain tumor with no current curative therapy. We found expression of the bZip transcription factor ATF5 in all 29 human glioblastomas and eight human and rat glioma cell lines assessed. ATF5 is not detectably expressed by mature brain neurons and astrocytes, but is expressed by reactive astrocytes. Interference with ATF5 function or expression in all glioma cell lines tested causes marked apoptotic cell death. In contrast, such manipulations do not affect survival of ATF5-expressing cultured astrocytes or of several other cell types that express this protein. In a proof-of-principle experiment, retroviral delivery of a function-blocking mutant form of ATF5 into a rat glioma model evokes death of the infected tumor cells, but not of infected brain cells outside the tumors. The widespread expression of ATF5 in glioblastomas and the selective effect of interference with ATF5 function/expression on their survival suggest that ATF5 may be an attractive target for therapeutic intervention in such tumors.

Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse.

Receptor protein tyrosine phosphatases (RPTPs) comprise a family of proteins that feature intracellular phosphatase domains and an ectodomain with putative ligand-binding motifs. Several RPTPs are expressed in the brain, including RPTP-kappa which participates in homophilic cell-cell interactions in vitro [Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, J. Sap, Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region, Mol. Cell. Biol. 13 (1993) 2942-2951; J. Sap, Y.-P. Jiang, D. Friedlander, M. Grumet, J. Schlessinger, Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding, Mol. Cell. Biol. 14 (1994) 1-9]. The homology of RPTP-kappa's ectodomain to neural cell adhesion molecules indicates potential roles in developmental processes such as axonal growth and target recognition, as has been demonstrated for certain Drosophila RPTPs. The brain distribution of RPTP-kappa-expressing cells has not been determined, however. In a gene-trap mouse model with a beta-gal+neo (beta-geo) insertion in the endogenous RPTP-kappa gene, the consequent loss of RPTP-kappa's enzymatic activity does not produce any obvious phenotypic defects [W.C. Skarnes, J.E. Moss, S.M. Hurtley, R.S.P. Beddington, Capturing genes encoding membrane and secreted proteins important for mouse development, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6592-6596]. Nevertheless, since the transgene's expression is driven by the endogenous RPTP-kappa promoter, distribution of the truncated RPTP-kappa/beta-geo fusion protein should reflect the regional and cellular expression of wild-type RPTP-kappa, and thus may identify sites where RPTP-kappa is important. Towards that goal, we have used this mouse model to map the distribution of the truncated RPTP-kappa/beta-geo fusion protein in the adult mouse brain using beta-galactosidase as a marker enzyme. Visualization of the beta-galactosidase activity revealed a non-random pattern of expression, and identified cells throughout the CNS that display RPTP-kappa promoter activity. Several neural systems highly expressed the transgene-most notably cortical, olfactory, hippocampal, hypothalamic, amygdaloid and visual structures. These well-characterized brain regions may provide a basis for future studies of RPTP-kappa function.

GGF/neuregulin induces a phenotypic reversion of oligodendrocytes.

We have previously shown that glial growth factor (GGF), a member of the neuregulin (NRG) family of growth factors, is a mitogen and survival factor for oligodendrocyte progenitors in cell culture and blocks their differentiation at the pro-oligodendrocyte stage (P. D. Canoll et al., 1996, Neuron 17, 229-243). We now show that GGF is able to induce differentiated oligodendrocytes to undergo a phenotypic reversion characterized by loss of MBP expression, reexpression of the intermediate filament protein nestin, reorganization of the actin cytoskeleton, and a dramatic reduction in the number of processes per cell. TUNEL analysis demonstrates that GGF is not cytotoxic for mature oligodendrocytes, but rather enhances their survival. GGF also induces the rapid activation of the PI 3-kinase and MAP kinase signaling pathways. These results further support a role for the NRGs in promoting the proliferation and survival of and inhibiting the differentiation of cells in the oligodendrocyte lineage and demonstrate that oligodendrocytes that differentiate in culture retain a substantial degree of phenotypic plasticity.

GGF/neuregulin is a neuronal signal that promotes the proliferation and survival and inhibits the differentiation of oligodendrocyte progenitors.

We show that GGF/neuregulin is a mitogen for prooligodendrocytes (O4+/O1- cells), oligodendrocytes (O4+/O1+ cells), and type-2 astrocytes. Heregulin beta 1, another neuregulin isoform, is also mitogenic. The proliferative effect of glial growth factor (GGF) does not require, but is greatly potentiated by, serum factors. GGF also promotes the survival of pro-oligodendrocytes under serum-free conditions. High levels of GGF reversibly inhibit the differentiation and lineage commitment of oligodendrocyte progenitors and, in differentiated cultures, result in loss of O1 and myelin basic protein expression. All three erbB receptors are expressed by progenitors and are activated by GGF; the relative abundance of these receptors changes during differentiation. Finally, cortical neurons release a soluble mitogen for pro-oligodendrocytes that is specifically blocked by antibodies to GGF. These results implicate the neuregulins in the neuronal regulation of oligodendrocyte progenitor proliferation, survival, and differentiation.

Three forms of RPTP-beta are differentially expressed during gliogenesis in the developing rat brain and during glial cell differentiation in culture.

In situ hybridization and Northern analysis demonstrate that the three splicing variants of RPTP-beta have different spatial and temporal patterns of expression in the developing brain. The 9.5-kb and 6.4-kb transcripts, which encode transmembrane protein tyrosine phosphatases with different extracellular domains, are predominantly expressed in glial progenitors located in the subventricular zone (SVZ). The 8.4-kb transcript, which encodes a secreted chondroitin sulfate proteoglycan (phosphacan), is expressed at high levels by more mature glia that have migrated out of the SVZ. The three transcripts are also differentially expressed in glial cell cultures; O2A progenitors express high levels of the 9.5- and 8.4-kb transcript, whereas type 1 astrocyte progenitors predominantly express the 6.4-kb transcript. C6 gliomas also express high levels of the 6.4-kb transcript. Treating C6 cells with the differentiating agent dibutyryl cyclic-AMP (DBcAMP), induces a decrease in the 6.4-kb transcript and a corresponding increase in the 8.4-kb transcript. O2A cells grown in the presence of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) remain highly proliferative and undifferentiated, and continue to express high levels of RPTP-beta. However, when O2A cells are grown in conditions that induce oligodendrocyte differentiation, there is a marked decrease in the expression of the transmembrane forms of RPTP-beta, as determined by immunofluorescence. These results demonstrate that RPTP-beta expression is regulated during glial cell differentiation and suggest that the different forms of RPTP-beta perform distinct functions during brain development.

Expression of receptor protein tyrosine phosphatase-sigma (RPTP-sigma) in the nervous system of the developing and adult rat.

The expression of receptor protein tyrosine phosphatase-sigma (RPTP-sigma) mRNA during rat development was examined by Northern blot and in situ hybridization analyses. Northern blot analysis revealed that the two transcripts (5.7 kb and 6.9 kb) had different spatial and temporal patterns of expression. The 6.9-kb transcript was more abundant during embryonic development, whereas the 5.7-kb transcript was more abundant during postnatal development and in the adult. In situ hybridization revealed that RPTP-sigma mRNA was widely expressed throughout the central and peripheral nervous system during embryonic development. Very high levels were seen in the ventricular zone, subventricular zone, cortex, dorsal root ganglia, cranial nerve ganglia, olfactory epithelium, and retina. During postnatal development the level of expression decreased in most brain regions. However, high levels continued to be seen in the hippocampus. Emulsion autoradiography revealed that the majority of RPTP-sigma mRNA is expressed in neurons. Northern analysis showed that cultured glial cells expressed the 6.9-kb transcript, but not the 5.7-kb. RPTP-sigma mRNA expression profiles were clearly distinct from those of leukocyte antigen-related protein (LAR), a closely related RPTP. The spatiotemporal pattern of RPTP-sigma mRNA expression indicates that RPTP-sigma may play a role in the development of the nervous system.

Cloning of neurotrimin defines a new subfamily of differentially expressed neural cell adhesion molecules.

Previous studies in the laboratory indicated that glycosylphosphatidylinositol (GPI)-anchored proteins may generate diversity of the cell surface of different neuronal populations (Rosen et al., 1992). In this study, we have extended these findings and surveyed the expression of GPI-anchored proteins in the developing rat CNS. In addition to several well characterized GPI-anchored cell adhesion molecules (CAMs), we detected an unidentified broad band of 65 kDa that is the earliest and most abundantly expressed GPI-anchored species in the rat CNS. Purification of this protein band revealed that it is comprised of several related proteins that define a novel subfamily of immunoglobulin-like (Ig) CAMs. One of these proteins is the opiate binding-cell adhesion molecule (OBCAM). We have isolated a cDNA encoding a second member of this family, that we have termed neurotrimin, and present evidence for the existence of additional family members. Like OBCAM, with which it shares extensive sequence identity, neurotrimin contains three immunoglobulin-like domains. Both proteins are encoded by distinct genes that may be clustered on the proximal end of mouse chromosome 9. Characterization of the expression of neurotrimin and OBCAM in the developing CNS by in situ hybridization reveals that these proteins are differentially expressed during development. Neurotrimin is expressed at high levels in several developing projection systems: in neurons of the thalamus, subplate, and lower cortical laminae in the forebrain and in the pontine nucleus, cerebellar granule cells, and Purkinje cells in the hindbrain. Neurotrimin is also expressed at high levels in the olfactory bulb, neural retina, dorsal root ganglia, spinal cord, and in a graded distribution in the basal ganglia and hippocampus. OBCAM has a much more restricted distribution, being expressed at high levels principally in the cortical plate and hippocampus. These results suggest that these proteins, together with other members of this family, provide diversity to the surfaces of different neuronal populations that could be important in the specification of neuronal connectivity.

The expression of a novel receptor-type tyrosine phosphatase suggests a role in morphogenesis and plasticity of the nervous system.

Analysis of the localization of receptor-type protein tyrosine phosphatase-beta (RPTP-beta) by in situ hybridization and immunocytochemistry indicates that it is predominantly expressed in the developing central nervous system (CNS). RPTP-beta is highly expressed in radial glia and other forms of glial cells that play an important role during development. The immunoreactivity localizes to the radial processes of these cells, which act as guides during neuronal migration and axonal elongation. The pattern of RPTP-beta expression changes with the progression of glial cell differentiation. In the adult, high levels of RPTP-beta are seen in regions of the brain where there is continued neurogenesis and neurite outgrowth. The spatial and temporal patterns of RPTP-beta expression suggest that this receptor phosphatase plays a role in morphogenesis and plasticity of the nervous system.

The cloning of a receptor-type protein tyrosine phosphatase expressed in the central nervous system.

We have isolated cDNA clones and deduced the complete amino acid sequence of a large receptor-type protein tyrosine phosphatase containing 2307 amino acids. The human gene encoding this phosphatase, denoted RPTP beta (or PTP zeta), has been localized to chromosome 7q31-33. RPTP beta is composed of a large extracellular domain, a single transmembrane domain, and a cytoplasmic portion with two tandem catalytic domains. We have also cloned a variant of RPTP beta lacking 859 amino acids from the extracellular domain but with intact transmembrane and cytoplasmic domains. Interestingly, the amino-terminal region of the extracellular domain of RPTP beta contains a stretch of 266 amino acids with striking homology to the enzyme carbonic anhydrase. Immunoprecipitation experiments from a human neuroblastoma cell line indicate that the apparent molecular mass of the core and glycosylated forms of RPTP beta are approximately 250 and 300 kDa, respectively. Northern blot analysis shows that RPTP beta is strictly expressed in the central nervous system. In situ hybridization was used to further localize the expression to different regions of the adult brain including the Purkinje cell layer of the cerebellum, the dentate gyrus, and the subependymal layer of the anterior horn of the lateral ventricle. Hence, RPTP beta represents the first mammalian tyrosine phosphatase whose expression is restricted to the nervous system. The high level of expression of RPTP beta transcripts in the ventricular and subventricular zones of the embryonic mouse brain suggests the importance of this tyrosine phosphatase in the development of the central nervous system.

Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases.

The tyrosine phosphatase RPTP gamma is a candidate tumor suppressor gene since it is located on human chromosome 3p14.2-p21 in a region frequently deleted in certain types of renal and lung carcinomas. In order to evaluate its oncogenic potential and to explore its normal in vivo functions, we have isolated cDNAs and deduced the complete sequences of both human and murine RPTP gamma. The murine RPTP gamma gene has been localized to chromosome 14 to a region syntenic to the location of the human gene. Northern (RNA) blot analysis reveals the presence of two major transcripts of 5.5 and 8.5 kb in a variety of murine tissues. In situ hybridization analysis reveals that RPTP gamma mRNA is expressed in specific regions of the brain and that the localization of RPTP gamma changes during brain development. RPTP gamma is composed of a putative extracellular domain, a single transmembrane domain, and a cytoplasmic portion with two tandem catalytic tyrosine phosphatase domains. The extracellular domain contains a stretch of 266 amino acids with striking homology to the zinc-containing enzyme carbonic anhydrase (CAH), indicating that RPTP gamma and RPTP beta (HPTP zeta) represent a subfamily of receptor tyrosine phosphatases. We have constructed a model for the CAH-like domain of RPTP gamma based upon the crystal structure of CAH. It appears that 11 of the 19 residues that form the active site of CAH are conserved in RPTP gamma. Yet only one of the three His residues that ligate the zinc atom and are required for catalytic activity is conserved. On the basis of this model we propose that the CAH-like domain of RPTP gamma may have a function other than catalysis of hydration of metabolic CO2.

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of [3H]dextromethorphan and sigma ligands to guinea pig brain.

The DM1/sigma 1 site binds dextromethorphan (DM) and sigma receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of [3H]dextromethorphan, [3H]3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine and (+)-[3H]1,3-Di-o-tolyl-guanidine ([3H]DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM Ki values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM1/sigma 1 site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed Ki values of 9-13 and 3-4 microM respectively against the three labeled ligands. These results, the broad specificity of the DM1/sigma 1 binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor. These findings may have important implications for the understanding of the therapeutic, side effects and toxicity of several neurotropic drugs.

Ropizine concurrently enhances and inhibits [3H]dextromethorphan binding to different structures of the guinea pig brain: autoradiographic evidence for multiple binding sites.

Ropizine (10 microM) produces a simultaneous enhancement and inhibition of [3H]dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [3H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhanced [3H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)-pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects of ropizine are due, at least in part, to the effects of ropizine on two different types of [3H]DM binding sites. However, this study does not rule out that common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.

Dextromethorphan sites, sigma receptors, and the psychotomimetic effects of sigma opiates.

A review of the literature shows that the dysphoric and psychotomimetic side effects of opiate agonist-antagonist reside in the opiate levorotatory isomer. Thus, the current definition of the sigma receptor cannot explain the psychotomimetic effects of sigma opiates.

Autoradiographic localization of [3H]dextromethorphan in guinea pig brain: allosteric enhancement by ropizine.

Dextromethorphan (DM) is an antitussive with anticonvulsant activity that binds to high- and low-affinity sites in guinea pig brain homogenates. We examined the autoradiographic localization of [3H]DM using the anticonvulsant ropizine, an allosteric modifier that decreases the dissociation rate of [3H]DM. Competition studies demonstrated that the binding to brain sections was identical to that of brain homogenates [Craviso and Musacchio: Mol Pharmacol 23:629-640, 1983b]. Computer-assisted quantitative analysis of the autoradiographic images demonstrated that [3H]DM binds to discrete structures throughout the brain, but with higher density in the midbrain, pons, and medulla oblongata. The most intense labeling was observed in the rhabdoid, dorsal raphe, median raphe, caudal linear raphe nuclei, and cranial motor nerve nuclei. The central gray showed moderate to high-density labeling throughout its entire rostro-caudal extent, with very high binding in the dorsal tegmental nucleus and the locus coeruleus. Moderate and high binding was also seen in several hypothalamic structures. Distinct bands of moderate binding were seen in the pyramidal cell layer of the piriform cortex, the retrosplenial cortex, the granular cell layer of the dentate gyrus, the pyramidal cell layer of the hippocampus, and the Purkinje cell layer of the cerebellum. The striking similarity between the binding distribution of [3H]DM and sigma ligands, plus competition studies in brain homogenate, support the hypothesis that DM and sigma ligands share a common high-affinity binding site [Musacchio et al: Mol Pharmacol 35:1-5, 1989]. The distribution of [3H]DM binding provides possible anatomical substrates for both the antitussive and anticonvulsant actions of DM.

Dextromethorphan and sigma ligands: common sites but diverse effects.

There is increasing evidence that sigma ligands and dextromethorphan (DM) bind to at least one common high-affinity site. DM and other antitussives do not produce psychotomimetic effects. This suggested that sigma ligands may produce their characteristic effects through another site, and prompted us to review critically the literature on the side effects of sigma opiates. Contrary to what is generally accepted, the dysphoric and psychotomimetic side effects of sigma opiates are mediated by the levo-and not by the dextrorotatory isomers. Moreover, these effects are unequivocally naloxone-reversible. Therefore, the current version of the "sigma receptor", with high affinity for the dextrorotatory sigma opiates, cannot explain the psychotomimetic effects of the levorotatory enantiomers. Thus, neither the "sigma ligands" nor its newly defined "receptor" are involved in the psychotomimetic effects of sigma opiates. Further experimentation with more selective drugs and with a combination of different methods will be necessary to identify the different binding sites, and to establish their physiological role and therapeutic potential.