Altered sensitivity of CD81-deficient mice to neurobehavioral effects of cocaine.

CD81, also known as target of the antiproliferative antibody, is known to be expressed in astrocytes and involved in cell adhesion and, recently, we demonstrated its induction exclusively in the accumbens following cocaine. In the present study, the sensitivity of CD81-deficient mice to behavioral effects of cocaine was evaluated. It was found that CD81-deficient mice exhibited altered sensitivity to cocaine as assessed in the place preference conditioning paradigm and locomotor activity. This deficit in place preference conditioning was not accompanied by a deficit in acquisition or retention of water maze behavior. In addition, CD81 knockout mice exhibited higher levels of nucleus accumbens dopamine as compared to their controls. These observations are discussed in the context of the role of CD81 in cocaine-mediated behaviors.

Immunohistochemical localization of EphA5 in the adult human central nervous system.

To better understand the functional role of EphA5 in the adult human central nervous system (CNS), we performed an immunohistochemical mapping study. EphA5, like other members of the Elk/Eph family of receptor tyrosine kinases, was widely distributed in CNS neurons. However, the distribution of the neuronal staining was not uniform. The abundance of stained neurons appeared to increase from the forebrain to the hindbrain and spinal cord. Glial and endothelial tissue was unstained. These findings are consistent with the existence of receptor and ligand gradients in different brain regions. The localization of EphA5 to motor and sensory neurons is consistent with a role of EphA5 in neural plasticity, cell-cell recognition, and topographical orientation of neuronal systems.

Functional activation of EphA5 receptor does not promote cell proliferation in the aberrant EphA5 expressing human glioblastoma U-118 MG cell line.

Eph receptors are a subfamily of receptor tyrosine kinases (RTKs), that are activated by ephrin ligands and appear to play important roles in axon guidance and cell migration during development of the nervous system. Over-expression or constitutive activation of Eph receptors has been linked with increased proliferation in various tumours. We have recently described lineage aberrant expression of EphA5 in primary human astrocytomas, glioblastomas and in the human glioblastoma U-118 MG cell line. A role for EphA5 expression in these tumours is not apparent, and we have investigated the cellular effects of EphA5 activation using the human glioblastoma U-118 MG cell line as a model. Immunofluorescent staining demonstrated cell surface expression of EphA5. Activation of the EphA5 receptor using an ephrin-A1 recombinant fusion protein resulted in tyrosine phosphorylation of EphA5 in a time-dependent manner. Exposure of U-118 MG glioblastoma cells to ephrin-A1 did not result in significant spontaneous or FCS-stimulated cell proliferation, though a marginal decrease was observed. This is in converse to the effects of Eph activation in other tumour cell lines, and is the first study to investigate EphA5 in glioblastoma cell lines.

Extensive splice variation and localization of the EHK-1 receptor tyrosine kinase in adult human brain and glial tumors.

EHK-1 is a neuronal ELK-related receptor tyrosine kinase which interacts with multiple, membrane-anchored ligands. Recent experiments have suggested a role for some of these ligands in the formation of neuronal pathways. Here, we report the isolation of human EHK-1 cDNAs and the localization of the human EHK-1 gene to chromosome 4q12. Six EHK-1 mRNA splice variants encoding cell-surface receptors with catalytic domains were identified in adult human brain where a 120-kDa EHK-1 protein predominates. Immunohistochemistry for EHK-1 reveals a dendritic staining pattern in cortical neurons and cerebellar Purkinje cells and a marked accumulation of EHK-1 in the somas of pyramidal neurons within the cortex and hippocampus. Interestingly, we have identified lineage aberrant expression of EHK-1 in a number of human gliomas. In addition to functions during development, EHK-1 may be involved in the maintenance of the adult nervous system and contribute to glioma development.

Reciprocal expression of myelin-associated glycoprotein splice variants in the adult human peripheral and central nervous systems.

The L- and S-MAG isoforms differ only at their C-terminus and are believed to be functionally distinct. To obtain information on the relative expression of these alternatively spliced isoforms in humans, we cloned an S-MAG cDNA fragment. The deduced amino-acid sequence of the human S-MAG C-terminus shows fairly conservative substitutions of 4 out of the 10 residues compared to the rodent peptide. Using reverse transcription and a competitive polymerase chain reaction, we show that, in contrast to rodents, the L-MAG splice variant predominates in adult human brain while, like in rodents, S-MAG transcripts are most abundant in peripheral nerve. The results obtained by Western blot analysis and immunohistochemistry are in good agreement with the findings at the mRNA level. Animal experiments may thus be more representative for the role of MAG in human nerve than in brain.

Selective loss of myelin-associated glycoprotein from myelin correlates with anti-MAG antibody titre in demyelinating paraproteinaemic polyneuropathy.

The IgM monoclonal autoantibodies of patients with demyelinating paraproteinaemic polyneuropathy recognize a carbohydrate structure present on both myelin-associated glycoprotein (MAG) and protein zero (P0). These autoantibodies are sufficient to cause the disease but the mechanism of demyelination remains unclear. We have analysed nerve biopsies from eight patients with polyneuropathy and anti-MAG antibodies by quantitative immunohistochemistry and find a concordant pattern of reduced expression of myelin markers with the loss of myelinated fibres. We report here novel features of this disease, in particular a selective lack of detectable MAG in a large proportion of myelinated-fibres containing P0, myelin basic protein (MBP) and periaxin. There is also an inverse correlation of the distribution of MAG in peripheral nerve myelin with the serum anti-MAG antibody titres but no correlation of these titres with the loss of myelinated fibres. Double immunofluorescence staining of paraproteinaemic polyneuropathy (PPN) nerves shows anti-MAG IgM deposited on the periphery of myelinated fibres associated with or lacking MAG staining. These data suggest that the binding of anti-MAG antibodies to MAG and/or other myelin component(s) results in MAG downregulation and may have an essential role in the molecular mechanisms leading to demyelination and partial regeneration in this disease.

Paraproteinaemic neuropathies.

Paraproteinaemia and neuropathy are each relatively frequent and may be associated by chance. However, a number of significant relationships have to be ruled out in the differential diagnosis. Malignant gammopathy should be excluded: multiple myeloma can lead to compression of the spinal cord or cauda equina; primary amyloidosis is occasionally involved; the rare but intriguing POEMS syndrome, consisting of polyneuropathy, organomegaly, endocrinopathy, M-protein and skin changes, usually accompanies osteosclerotic myeloma. It can be associated with angio-follicular lymph node hyperplasia and needs to be recognized because radioablative therapy is curative. The 'benign' monoclonal gammopathies of undetermined significance, known as MGUS, are much more frequent. There is an IgM MGUS group with predominantly distal sensorimotor demyelinating polyneuropathy and another rather heterogeneous group with IgG or IgA MGUS and a tendency to a favourable response to plasmapheresis. The role of the monoclonal IgG and IgA antibodies is unclear. This chapter has focused on the pathogenetic mechanisms of neuropathies associated with IgM MGUS. In the majority of cases, monoclonal autoantibodies specific for particular carbohydrate epitopes bind to myelin and are now recognized as the primary cause of the disease manifestations, including widening of the myelin lamellae. While the autoantibodies have been shown to bind complement, the presence of inhibitors is invoked to explain the absence of acute inflammatory changes. The epitopes recognized with the highest affinity by the auto-antibodies are present on the myelin-associated glycoprotein (MAG) and could interfere with cell adhesion and cellular signally processes. In addition, binding to antigenically similar glycoproteins, such as PO, PMP-22 and some acidic glycolipids, may be a contributory factor. It is generally accepted that the anti-MAG autoantibodies are inducing a progressive demyelinating polyneuropathy by modifying axon-Schwann cell interactions.

Recognition of human recombinant myelin associated glycoprotein by anti-carbohydrate antibodies of the L2/HNK-1 family.

The L2 and HNK-1 monoclonal antibodies recognize carbohydrate determinants containing sulfate-3-glucuronate that are prominent on cells of neural crest lineages. In humans these epitopes are most abundant on the Myelin Associated Glycoprotein and it was assumed that they co-localize on the same molecules. Recently, in vitro synthesized carbohydrates have provided a basis for the different recognition requirements of these two antibodies. We now provide in vivo evidence that a human melanoma cell line can produce glycoproteins such as fibronectin, which is recognized by both the L2 and HNK-1 antibodies, and simultaneously a transfected Myelin Associated Glycoprotein carrying only L2-type carbohydrates. Conceivably, the differential expression of L2- and HNK-1 type glycans could have a role in development.

Expression and developmental regulation of Ehk-1, a neuronal Elk-like receptor tyrosine kinase in brain.

Protein tyrosine kinases are pivotal in central nervous tissue development and maintenance. Here we focus on the expression of Ehk-1, a novel Elk-related receptor tyrosine kinase. Ehk-1 gene expression is observed in the developing and adult central nervous system and is highly regulated throughout development at both the messenger RNA and protein levels. Three messenger RNA transcripts of 8.5, 5.9 and 5.1 kb are detectable in the rat brain and a variety of splice possibilities have been identified. However, a major protein species of around M(r) 120,000 predominates throughout development. Ehk-1 messenger RNA and protein levels are highest in the first postnatal week. By in situ messenger RNA hybridization the gene is expressed by all neurons of the adult brain, but mostly in the hippocampus, cerebral cortex and large neurons of the deep cerebellar nuclei, as well as the Purkinje and granular cells of the cerebellum. At earlier stages of development, transcripts are most prominent in the periventricular germinal layers of the brain. Immunohistochemistry reveals a pronounced membrane associated protein expression in immature neurons. In the adult animal, peak reactivity was found in the neuropil with sparing of most perikarya. The spatial and temporal pattern of ehk-1 gene expression suggests a role in both the development and maintenance of differentiated neurons of the central nervous system.

Altered growth and phenotype in clonal mycN transfectants of the SK-N-SH neuroblastoma cell line.

We have attempted to distinguish in human neuroblastoma between the effects of mycN on differentiation and its potential to promote malignant progression. Others have observed out-growth of autocrine cells with evidence of an advanced malignant phenotype in a mycN-transfected clonal cell line derived from the single-copy mycN neuroblastoma, SK-N-SH. We have now transfected the parental cell line with the same mycN expression vector and selected 5 clones characterized by unique and stable chromosomal integration sites and variable exogenous copy numbers. mycN gene expression was variable in the different clones and correlated roughly with the copy number of transfected mycN genes. Clones with minimal levels of mycN gene expression had a neuroblastic phenotype and low numbers of surface HLA class-I molecules. Clones with high levels of mycN expression had a Schwann/glial-like phenotype with higher surface HLA class-I display without imbalance of expression of specific loci and accelerated growth. Two such clones were capable of anchorage-independent growth in the absence of serum, and acquired tumorigenic properties. Our results show that exogenous mycN expression can be associated with a differentiation of neuroblastoma cells along the Schwann/glial pathway and can induce accelerated and autonomous growth.

Characterization of a novel murine testis-specific serine/threonine kinase.

Using degenerate oligos corresponding to two highly conserved motifs within the protein kinase catalytic domain and a PCR-based cloning strategy, we have isolated a cDNA fragment encoding a new member of the Ser/Thr (serine/threonine) family of protein kinases. Expression analysis revealed that the fragment recognized two transcripts (1.6 and 1.4 kb) exclusively in testis. Using this fragment as a probe, we have cloned a full-length cDNA from a mouse testis cDNA library. The sequence has a 1092-bp open reading frame encoding a protein of 364 amino acids. The N-terminally localized kinase catalytic domain has all the conserved motifs found in other Ser/Thr kinases. Northern blot analysis using the full-length sequence as a probe revealed that the cloned gene corresponds to the 1.6-kb transcript, suggesting the existence of at least two testis-specific novel Ser/Thr kinases. We propose the name testis-specific kinase-1 (TSK-1) for the gene described here. A GenEMBL databank search revealed highest homology to the human gene encoding rac protein kinase-beta and the group of yeast Ser/Thr kinases encoded by SNF-1, nim-1, KIN-1 and KIN-2.

Binding of myelin basic protein peptides to human histocompatibility leukocyte antigen class II molecules and their recognition by T cells from multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune disease in which myelin proteins have been implicated as autoantigens recognized by pathogenic autoreactive T cells. To study the relationship between human myelin basic protein (hMBP) and HLA alleles associated to MS susceptibility, such as DRB1*1501, the binding of synthetic peptides spanning the entire hMBP sequence to 10 purified HLA-DR molecules was determined. All the hMBP peptides tested showed binding affinity for at least one of the DR molecules analyzed, but three hMBP peptides, included in sequences 13-32, 84-103, and 144-163 were found capable of binding to three or more DR molecules. The hMBP peptide 84-103 was the most degenerate in binding, in that it bound to 9 out of 10 DR molecules tested. Interestingly, it bound with highest affinity to DRB1*1501 molecules. To correlate the binding pattern of hMBP peptides to HLA class II molecules with their recognition by T cells, 61 hMBP-specific T cell lines (TCL) were established from the peripheral blood of 20 MS patients, who were homozygous, heterozygous, or negative for DRB1*1501. Analysis of hMBP epitopes recognized by these TCL and their HLA restriction demonstrated a very good correlation between binding data and T cell proliferation to hMBP peptides. Although virtually all hMBP peptides tested could be recognized by at least one TCL from MS patients, three immunodominant T cell epitopes were apparent among the TCL examined, corresponding exactly to the hMBP peptides capable of binding to several DR molecules. No major difference could be detected in the recognition of immunodominant hMBP peptides by TCL from DRB1*1501 positive or negative MS patients. These results have implications for the role of hMBP as relevant autoantigen, and of DRB1*1501 as susceptibility allele in MS.

Protein kinases expressed in aggregating brain cell cultures during myelination.

Serum-free aggregating rat brain cell cultures provide sufficient cell surface and paracrine interactions between neurons and glial cells for compact myelination. We are interested in the part played in these signalling pathways by protein kinases and have used a PCR cDNA cloning approach to catalogue the protein kinase genes expressed by these cultures. 8 transmembrane protein kinases were identified: IGF1-R, trk B, bFGF-R, c-met, Tyro2, Tyro1, Tyro4 and a novel eck-related gene. The first 4 are receptors for ligands with known trophic functions. Tyro2 is a novel gene related to the EGF-R. The latter 3 belong to the eck gene family of more than 8 highly related putative receptors for, as yet, unknown ligands. 8 cDNAs for intracellular protein kinases were also isolated including 3 novel genes. Ongoing studies are investigating whether these proteins contribute to myelination and/or could be used as therapeutic targets in demyelinating diseases.

Expression of the B cell-associated tyrosine kinase gene Lyn in primary neuroblastoma tumours and its modulation during the differentiation of neuroblastoma cell lines.

The src-related intracellular protein tyrosine kinase Lyn is a signal transducing molecule for surface immunoglobulin M and is expressed predominantly in hemopoietic cells. We report here the expression of the lyn gene in human neuroblastoma. In surgical tumour samples lyn transcripts were found preferentially at early stages whereas they were barely detectable in highly malignant tumours. In a cloned human neuroblastoma cell line, Be(2)C, lyn mRNA levels increased during neuronal differentiation induced by retinoic acid. Lyn mRNA levels were undetectable and did not respond to retinoic acid in a glial-type neuroblastoma clone, SH-EP. Retinoic acid-induced glial differentiation was associated with a reduction of lyn transcripts in a clonal I-type neuroblastoma cell line, SH-IN, which shares properties of both neuronal- and glial-type clones. Like pp60c-src Lyn may be involved in a signalling pathway of neuroblasts committed to neuronal differentiation.

T cell receptor (TcR) beta chain transgenic mice: studies on allelic exclusion and on the TcR+ gamma/delta population.

To study allelic exclusion of TcR genes we analyzed two types (I and II) of TcR beta transgenic mice. T cells derived from both types of mice contained similar amounts of transgenic RNA transcripts; however, surface expression of the transgenic beta chain was drastically reduced in type II compared to type I. In type I transgenic mice, productive rearrangements and expression of endogenous TcR beta genes were suppressed whereas on T cells of type II mice, both transgenic and endogenous TcR beta chains were expressed on the surface of the same cell. These findings suggest that allelic exclusion of TcR genes in beta transgenic mice depends on amount and/or onset of transgene expression during thymic development. Furthermore, TcR gamma rearrangements and the population of TcR gamma/delta-bearing double-negative CD4-CD8- thymocytes were reduced fivefold in type I transgenic animals. However, the V gamma usage and the gamma/delta+ dendritic epidermal cell populations appeared normal. RNase protection analysis further revealed low levels of transgenic TcR beta chain transcripts in TcR+ gamma/delta CD4-CD8- thymocytes. These results suggest that the beta transgene only quantitatively influences the gamma/delta T cell compartment, and supports the independence of the gamma/delta population.

Selective expression of V delta 6 genes by B2A2- CD4- CD8- T cell receptor gamma/delta thymocytes.

Most CD4-CD8- adult murine thymocytes characterized by absence of the B2A2 (J11d) antigen express T cell receptors (TcR) alpha/beta and utilize preferentially V beta 8.2 segments. To a much lesser extent, B2A2-CD4-CD8- thymocytes also express TcR gamma/delta, as evidenced by biochemical and Northern blot analysis. We have now been able to exclude the possibility that these cells might co-express both types of TcR: V beta 8+ B2A2- CD4-CD8- thymocytes expressed no TcR delta mRNA whereas the corresponding V beta 8- subset transcribed full-length TcR gamma as well as delta mRNA. Furthermore, the TcR gamma/delta expressing B2A2- thymocytes were found to use preferentially V delta 6 genes. Conversely, they did not express V delta 5 genes which are most frequently used by other TcR gamma/delta-bearing populations such as B2A2+ CD4-CD8- thymocytes or CD4-CD8- peripheral lymph node cells. RNase protection experiments showed that two particular V delta 6 transcripts predominate in these gamma/delta populations, the most prominent V delta 6 sequence being highly homologous if not identical to V alpha 7.1. Our observations extend previous information on overlapping V alpha and V delta gene repertoires, particularly in the cross-hybridizing V alpha 7/V delta 6 gene family. Moreover, our data suggest that B2A2-CD4-CD8- thymocytes represent a developmentally unique subset in which both V delta and V beta segments are nonrandomly expressed.

Production and characterization of a rat monoclonal antibody against the murine CD3 molecular complex.

We describe the production of a rat monoclonal antibody, 17A2, that detects the T cell receptor-associated CD3 molecular complex. 17A2 cross-competes with a CD3 epsilon-specific reagent and similarly stimulates IL-2 production by T cells. Immunohistological and cell separation applications are shown.

CD3-associated alpha/beta and gamma/delta heterodimeric receptors are expressed by distinct populations of CD4- CD8- thymocytes.

Immature double negative (DN) CD4-8- thymocytes expressing the CD3 molecular complex can be subdivided into two distinct subsets based on expression of the B2A2 antigen. Thus, B2A2+ CD3+ DN thymocytes express CD3-associated 35 kDa and 45 kDa polypeptide chains characteristic of a gamma/delta T cell receptor, whereas B2A2- DN thymocytes express predominantly 38 to 43 kDa CD3-associated structures typical of alpha/beta TCR. At the mRNA level, B2A2+ DN thymocytes express full length gamma and delta transcripts but no alpha message and only short (1 kb) B transcripts. In contrast, the B2A2- DN subset expresses full length alpha and beta transcripts and minimal levels of delta transcripts. Interestingly, B2A2- DN thymocytes, unlike mature T cells, also express abundant levels of full length TCR gamma mRNA, perhaps suggesting that these cells represent an early stage after divergence of the gamma/delta and alpha/beta T cell lineages.