Clozapine increases apolipoprotein D expression in rodent brain: towards a mechanism for neuroleptic pharmacotherapy.

In contrast to typical neuroleptic drugs, which have high affinities for dopamine D2 receptors, clozapine binds to multiple neurotransmitter receptors. The mechanisms responsible for its superior clinical efficacy over typical neuroleptics remain unknown. Using an automated genomics approach, total gene expression analysis (TOGA), we found an approximately threefold increase in the accumulation of the mRNA encoding apolipoprotein D (apoD) in mouse striatum in response to chronic treatment with clozapine. While in control animals, apoD is expressed predominantly in astrocytes, in situ hybridization and immunohistochemical studies indicated a substantial increase in apoD expression in neurons of the striatum, globus pallidus and thalamus after 2 weeks of clozapine treatment. Clozapine-induced increases in apoD expression were also observed in some white matter regions. These results suggest that apoD is a mediator in the mechanisms of clozapine and thus that deficiencies in aspects of lipid metabolism may be responsible for psychoses.

Identification of genes in the oligodendrocyte lineage through the analysis of conditionally immortalized cell lines.

The mouse oligodendrocyte cell lines, N19 and N20.1, were used as sources of potential stage-specific RNA in order to construct a subtraction library enriched in cDNAs expressed early in the oligodendrocyte (OL) lineage. From this library, 23 clones were examined and three were examined in most detail. The mRNAs of the three library clones were preferentially expressed in the N19 (progenitor) compared to the N20.1 (immature) OL line. One of these corresponded to the intermediate filament protein cytokeratin K19, which has not been reported to be expressed in OLs previously. Another was identified as the mouse homolog of T-cadherin, previously reported not to be present in OLs. Antisera raised against a T-cadherin peptide indicated the protein colocalized with the OL lineage markers A(2)B(5), A007, and 01 in mouse primary glial cultures. However, small round cells resembling OL precursors labeled intensely with T-cadherin, but were negative for the other markers, suggesting that this gene might be expressed earlier in the lineage. In early postnatal brain, in addition to the expected neuronal tracts, the T-cadherin antibody labeled small bipolar cells, approximately 8-10 microm in diameter, in white matter tracts. These cells had the morphology of OLs or their precursors and were identified within the cerebellar white matter and the corpus callosum, regions rich in OLs. The third clone, 3g5, was homologous to the P8 clone isolated from rat pancreas. It encoded an 80-amino-acid polypeptide with a protein kinase C domain suggesting a possible role in signal transduction. Antisera to this peptide also colocalized 3g5 with cells expressing A(2)B(5), A007, and 01 in culture and in cells within white matter tracts which had the same morphology as those labeled by T-cadherin in these regions. In addition to these, beta(10) thymosin and mevalonate kinase clones were also isolated from the screen.

Embryonic expression of the myelin basic protein gene: identification of a promoter region that targets transgene expression to pioneer neurons.

The myelin basic protein (MBP) gene produces two families of structurally related proteins from three different promoters-the golli products, generated from the most upstream promoter, and the MBPs, produced from the two downstream promoters. In this report we describe the expression of golli proteins within some of the earliest neuronal populations of the brain, including Cajal-Retzius cells and preplate neurons of the forebrain, representing a new marker for these cells. To identify elements responsible for neuronal expression of the golli products, we generated transgenic animals from constructs containing different portions of the upstream promoter. A construct containing 1.1 kb immediately upstream of the golli transcription start site targeted expression of beta-galactosidase to preplate neurons and a subset of Cajal-Retzius cells in transgenic mice-the first reported genetic element to target expression to these pioneer cortical populations. Although expression in Cajal-Retzius cells declined with embryonic development, preplate cells continued to express the transgene after arriving at their final destination in the subplate. Interestingly, expression persisted in subplate neurons found within a distinct layer between the white matter and cortical layer VI well into postnatal life. Birth dating studies with bromodeoxyuridine indicated that these neurons were born between E10.5 and E12.5. Thus, the transgene marked subplate neurons from their birth, providing a fate marker for these cells. This work suggests a role for the MBP gene in the early developing brain long before myelination and especially in the pioneer cortical neurons important in the formation of the cortical layers.

Myelin protein expression is increased in lymph nodes of mice with relapsing experimental autoimmune encephalomyelitis.

Myelin proteins had been thought to be sequestered behind the blood-brain barrier. Recently, however, myelin proteins have been found to be expressed in lymphoid tissues. The myelin basic protein (MBP) gene is embedded within a larger transcription unit called the golli-MBP gene. This larger gene encodes both the "classic" MBPs as well as the structurally related golli-MBPs. In this study, golli-MBP expression in lymph nodes was examined in four different models of relapsing experimental autoimmune encephalomyelitis (rEAE). Disease in these rEAE models was induced by the adoptive transfer of T lymphocytes specific for 18.5-kDa MBP, MBP peptide 83-102, or PLP peptide 139-151 in the SJL/J mouse and the adoptive transfer of T lymphocytes specific for MBP peptide Ac1-9 in the (SJL/J x PL/J)F1 mouse. In all four models, expression of golli-MBP BG21 mRNA was increased two- to fivefold in lymph nodes of mice 45 to 60 days post-transfer. Immunohistochemical analysis indicated that expression occurred principally in macrophages within lymph nodes. Endogenous golli-MBP epitopes within lymph node cells stimulated "classic" MBP 1-44-specific T lymphocytes, and this stimulatory ability resided within the adherent lymph node cell population. An increase in myelin protein expression within lymph nodes during rEAE has implications with regard to intra- and intermolecular epitope spreading. This is the first report describing an increase in target autoantigen expression within lymphoid tissue during an autoimmune disease.

Expression of the myelin basic protein gene locus in neurons and oligodendrocytes in the human fetal central nervous system.

The myelin basic protein (MBP) gene locus is composed of two overlapping transcription units that share all of the MBP exons. One of these transcription units expresses the MBPs and the other expresses a family of proteins structurally related to the MBPs. This second transcription unit is called the Golli gene, and the entire complex is called the Golli-mbp gene. In this study, the expression of the Golli gene was examined in the human fetal central nervous system (CNS). By using reverse transcriptase-polymerase chain reaction cloning we have identified eight new members of the Golli gene family of transcripts expressed in the human CNS. Golli gene expression was examined by in situ hybridization and immunohistochemistry, and surprisingly, Golli products were found to be expressed in neurons as well as oligodendrocytes. Furthermore, the subcellular distribution of Golli immunoreactivity in fetal spinal cord interneurons shifted between the various laminae. Golli protein was localized within the nuclei of interneurons in the posterior horn, but was found in the cell bodies and processes of interneurons in the anterior horn. Within oligodendrocytes, Golli protein was detected in the cell bodies and processes, including processes which were wrapping axonal segments. Golli mRNA expression was also observed in neurons within the cerebral cortex between 18 and 20 weeks postconception, prior to myelination of this brain region. During this period, there was a striking developmental increase in the numbers and in the locations of neurons expressing Golli mRNAs within the cortical plate. The diverse distribution of Golli proteins within neurons and oligodendrocytes indicates that their function is quite different from that of the MBPs to which they are closely related.

The major myelin protein genes are expressed in the human thymus.

Demyelinating diseases, such as multiple sclerosis (MS) in man or experimental allergic encephalomyelitis (EAE) in rodents, may include an associated immune response directed against myelin protein antigens such as the proteolipid protein (PLP) and the myelin basic protein (MBP). Development of an immune response has been attributed, in part, to the sequestration of central nervous system antigens behind the blood-brain barrier. Recently, we identified a novel gene, the golli gene, which overlaps the mbp gene. The Golli transcription unit produces a family of mRNAs, and their corresponding proteins possess MBP epitopes known to be encephalitogenic in EAE. Transcription of the golli gene was detected in immune system tissue. Therefore, we wished to determine whether genes that encode the two major myelin protein components, PLP and MBP, were expressed in the human thymus. Our data demonstrate that both the plp and golli genes are transcribed in the fetal human thymus. Moreover, both the PLP and DM-20 transcripts are produced from the plp gene, and the HOG 7 and HOG 5 transcripts are produced from the golli gene. Confocal fluorescent immunohistochemistry using antibodies for the PLP/DM-20 and Golli proteins, co-localized expression of these antigens to thymic macrophages. Thus, the plp and golli genes are expressed, and their corresponding protein produced, in an antigen presenting cell in the human immune system.

Expression of the myelin proteolipid protein gene in the human fetal thymus.

We have analyzed human fetal thymus and spleen for expression of the proteolipid protein (PLP) gene. We demonstrate that the PLP gene is transcribed in both tissues, and that both the PLP and DM-20 mRNAs are produced. Western blot analyses revealed that both the PLP and DM-20 protein isoforms were present in the fetal thymus and spleen. Immunohistochemical analyses indicated that the PLP/DM-20 proteins were detected in cells which have the distribution and morphology of thymic macrophages. These results provide further evidence that the PLP and DM-20 proteins are expressed in cell types other than myelin forming cells and possess function(s) unrelated to myelin structure. Furthermore, these data demonstrate that the PLP and DM-20 proteins are not shielded from the immune system behind the blood-brain barrier. These observations directly impinge upon the debate concerning acquisition of tolerance and the recognition that the encephalitogenic nature of PLP in diseases, such as Multiple Sclerosis, may not simply be related to its 'sequestration' from a 'naive' immune system.

Myelin basic protein gene expression in neurons: developmental and regional changes in protein targeting within neuronal nuclei, cell bodies, and processes.

The myelin basic protein (MBP) gene is part of the golli-mbp gene complex. In mouse, the golli-mbp gene produces two families of mRNAs from different transcription start sites that generate either MBPs or golli proteins (which contain MBP sequences in addition to unique peptide sequences). In situ hybridization and immunocytochemical analyses indicate that golli products are expressed in selected neuronal populations in postnatal mouse brain, in addition to oligodendrocytes, as shown earlier. The principal subcellular location of golli proteins in neurons was in axonal and dendritic processes. In a small subset of neurons, golli proteins were located in nuclei. With development and neuronal maturation, golli-mbp expression decreased and/or there was a striking shift in subcellular localization from nuclei and cell soma to the cell processes in specific neuronal populations. Golli protein was localize in neurites of migrating cerebellar granule cells, but it shifted to a nuclear localization when the cells took up residence in the internal granule cell layer. In some regions, (e.g., olfactory bulb and cerebellum) golli proteins were expressed over the entire postnatal period examined (birth to 75 d). The unique patterns of developmental expression within individual populations of neurons, and the unusual shift in subcellular localization of golli proteins with neuronal migration and maturation, suggest a complex regulation of this gene at both the transcriptional and posttranslational levels. The data also suggest that the cellular function(s) of the golli proteins is very different from the structurally related MBPs.

The human myelin basic protein gene is included within a 179-kilobase transcription unit: expression in the immune and central nervous systems.

Two human Golli (for gene expressed in the oligodendrocyte lineage)-MBP (for myelin basic protein) cDNAs have been isolated from a human oligodendroglioma cell line. Analysis of these cDNAs has enabled us to determine the entire structure of the human Golli-MBP gene. The Golli-MBP gene, which encompasses the MBP transcription unit, is approximately 179 kb in length and consists of 10 exons, seven of which constitute the MBP gene. The human Golli-MBP gene contains two transcription start sites, each of which gives rise to a family of alternatively spliced transcripts. At least two Golli-MBP transcripts, containing the first three exons of the gene and one or more MBP exons, are produced from the first transcription start site. The second family of transcripts contains only MBP exons and produces the well-known MBPs. In humans, RNA blot analysis revealed that Golli-MBP transcripts were expressed in fetal thymus, spleen, and human B-cell and macrophage cell lines, as well as in fetal spinal cord. These findings clearly link the expression of exons encoding the autoimmunogen/encephalitogen MBP in the central nervous system to cells and tissues of the immune system through normal expression of the Golli-MBP gene. They also establish that this genetic locus, which includes the MBP gene, is conserved among species, providing further evidence that the MBP transcription unit is an integral part of the Golli transcription unit and suggest that this structural arrangement is important for the genetic function and/or regulation of these genes.

Structure and developmental regulation of Golli-mbp, a 105-kilobase gene that encompasses the myelin basic protein gene and is expressed in cells in the oligodendrocyte lineage in the brain.

We have identified a novel transcription unit of 105 kilobases (called the Golli-mbp gene) that encompasses the mouse myelin basic protein (MBP) gene. Three unique exons within this gene are alternatively spliced into MBP exons and introns to produce a family of MBP gene-related mRNAs that are under individual developmental regulation. These mRNAs are temporally expressed within cells of the oligodendrocyte lineage at progressive stages of differentiation. Thus, the MBP gene is a part of a more complex gene structure, the products of which may play a role in oligodendrocyte differentiation prior to myelination. One Golli-mbp mRNA that encodes a protein antigenically related to MBP is also expressed in the spleen and other non-neural tissues.

Stable ring chromosome 21: molecular and clinical definition of the lesion.

Ring chromosome 21 results in deletions of chromosome 21. We report on a cytogenetic and molecular analysis of a 4-generation family segregating a stable ring chromosome 21 in 4 relatives. To investigate the molecular structure of the ring chromosome, we have analyzed the DNAs of the transmitted ring in a mother and her daughter. The daughter presented at the age of 2 years with severe growth retardation and microcephaly, whereas her mother had microcephaly but normal intelligence. High resolution chromosome analysis of both cases showed the ring chromosome to be r(21)(p13q22) resulting in deletions of 21p and 21q22. The molecular content of the ring chromosome was determined using quantitative Southern blot analyses of 5 random DNA sequences and 4 expressed genes assigned to chromosome 21 and mapping in the region of q22.3. We have shown that collagen type VI, alpha 2 (COL6A2,) S100 protein, beta polypeptide (neural), (S100B), and D21S44 are present in only one copy in both ring carriers, while CRYA1, CBS, D21S43, D21S42, D21S41, and D21S39 are present in two copies. These data and the breakpoints defining the deletion in these patients show that deletion of COL6A2 and S100B is compatible with normal function and confirm the physical map of 21q22.3 by placing COL6A2, S100B, and D21S44 in very distal 21q22.3. Patients with such small deletions provide unique models for understanding the biological and clinical significance of aneuploidy for specific expressed genes.

Transcription termination at the chicken beta H-globin gene.

We characterized the transcription termination region of the chicken beta H-globin gene. First we located the region by nuclear runon transcription in vitro. Then we sequenced and subcloned it into a chloramphenicol acetyltransferase (CAT) expression vector for assay in vivo. The region of beta H termination contains two interesting elements located about 1 kilobase downstream of the beta H gene poly(A) site. Either element alone can block CAT expression if inserted between the promoter and the poly(A) site of the cat gene in pRSVcat. The first element in the termination region is an unusually large inverted repeat in the DNA (delta G = -71 kcal). The second element, 200 base pairs further downstream, is an RNA polymerase II promoter which directs transcription back upstream on the complementary strand. This transcription converges on and collides with that from the beta H gene at or near the inverted repeat where transcription from both directions stops. We propose that the inverted repeat is a strong pause site which positions the converging polymerases for mutual site-specific termination.

Novobiocin induces the in vivo cleavage of active gene sequences in intact cells.

When the topoisomerase II inhibitor, novobiocin, is administered to embryonic chicken red blood cells, it induces the in vivo release of an endogenous nuclease which cleaves specifically within internucleosomal spacer DNA and within nuclease-hypersensitive sites in the active chromatin of intact cells. This in vivo released nuclease activity is induced by novobiocin only in metabolically active immature red blood cells. Little induction occurs in mature erythrocytes and no induction occurs in cells previously treated with 2,4-dinitrophenol. Although novobiocin is required to induce release and/or activation of the nuclease, the activity of the nuclease, once activated, is independent of novobiocin. Analysis of the cleaved DNA in drug-treated immature cells demonstrates that the novobiocin-induced nuclease has an unusual blunt-ended double-stranded mode of cleavage. Because of its special properties and apparent chromatin related function in vivo, the novobiocin-induced nuclease activity offers a novel and useful in vivo and in vitro probe of chromatin structure.

Hemoglobin switching in chickens. Is the switch initiated post-transcriptionally?

We have investigated the transcriptional specificity of chick embryo erythroid nuclei as a function of developmental age during progression of the hemoglobin switch. Nuclei were allowed to transcribe in vitro in the presence of high specific activity [alpha-32P]CTP and the radioactive transcripts were hybridized to a collection of plasmid clones spanning the beta-like globin gene region of the chicken genome. The results reveal locus-specific waves of transcription appearing during the interval between 5 and 12 days of incubation. The last wave, which comes in at 12 days, is highly specialized in transcription of the adult beta-globin gene locus. The most interesting wave of transcription occurs at 6-7 days. The most actively transcribed gene in the early part of this wave is the embryonic rho gene. As the wave progresses, the rho gene activity gradually gives way to beta gene transcription. Definitive red blood cells, which would be synthesizing little rho globin protein at this stage, appear to be responsible for the rho gene transcription. These results and additional data which we present indicate that during the initial stages of the hemoglobin switch the embryonic globin genes are silenced post-transcriptionally.