Transcriptional profiling reveals evidence for signaling and oligodendroglial abnormalities in the temporal cortex from patients with major depressive disorder.

Major depressive disorder is one of the most common and devastating psychiatric disorders. To identify candidate mechanisms for major depressive disorder, we compared gene expression in the temporal cortex from 12 patients with major depressive disorder and 14 matched controls using Affymetrix HgU95A microarrays. Significant expression changes were revealed in families of genes involved in neurodevelopment, signal transduction and cell communication. Among these, the expression of 17 genes related to oligodendrocyte function was significantly (P < 0.05, fold change > 1.4) decreased in patients with major depressive disorder. Eight of these 17 genes encode structural components of myelin (CNP, MAG, MAL, MOG, MOBP, PMP22, PLLP, PLP1). Five other genes encode enzymes involved in the synthesis of myelin constituents (ASPA, UGT8), or are essential in regulation of myelin formation (ENPP2, EDG2, TF, KLK6). One gene, that is, SOX10, encodes a transcription factor regulating other myelination-related genes. OLIG2 is a transcription factor present exclusively in oligodendrocytes and oligodendrocyte precursors. Another gene, ERBB3, is involved in oligodendrocyte differentiation. In addition to myelination-related genes, there were significant changes in multiple genes involved in axonal growth/synaptic function. These findings suggest that major depressive disorder may be associated with changes in cell communication and signal transduction mechanisms that contribute to abnormalities in oligodendroglia and synaptic function. Taken together with other studies, these findings indicate that major depressive disorder may share common oligodendroglial abnormalities with schizophrenia and bipolar disorder.

Application of cDNA microarrays to examine gene expression differences in schizophrenia.

Using cDNA microarrays we have investigated gene expression patterns in brain regions of patients with schizophrenia. A cDNA neuroarray, comprised of genes related to brain function, was used to screen pools of samples from the cerebellum and prefrontal cortex from a matched set of subjects, and middle temporal gyrus, from a separate subject cohort. Samples of cerebellum and prefrontal cortex from neuroleptic naive patients were also included. Genes that passed a 3% reproducibility criterion for differential expression in independent experiments included 21 genes for drug-treated patients and 5 genes for drug-naive patients. Of these 26 genes, 10 genes were increased and 16 were decreased. Many of the differentially expressed genes were related to synaptic signaling and proteolytic functions. A smaller number of these genes were also differentially expressed in the middle temporal gyrus. The five genes that were differentially expressed in two brain regions from separate cohorts are: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide; sialyltransferase; proteasome subunit, alpha type 1; ubiquitin carboxyl-terminal esterase L1; and solute carrier family 10, member 1. Identification of patterns of changes in gene expression may lead to a better understanding of the pathophysiology of schizophrenia disorders.

Levels of mRNAs encoding synaptic vesicle and synaptic plasma membrane proteins in the temporal cortex of elderly schizophrenic patients.

BACKGROUND: Electron microscopy and biochemical studies indicate that developmental abnormalities in synaptic organization may be present in brains of schizophrenic patients. This study determined whether these synaptic abnormalities are reflected in differential or uniform alterations in the expression of various synaptic protein genes in the left superior temporal gyrus of schizophrenic patients. METHODS: Levels of mRNAs encoding four synaptic vesicle proteins (synaptotagmin I [p65], rab3a, synaptobrevin 1, and synaptobrevin 2) and two synaptic plasma membrane proteins (syntaxin 1A and SNAP-25) were measured postmortem in the left superior temporal gyrus from elderly (58-95 years) schizophrenic patients (n = 14) and age-matched control subjects (n = 9). RESULTS: There were significant negative correlations between age and levels of synaptotagmin I (p65), rab3a, synaptobrevin 1, SNAP-25, and syntaxin 1A mRNAs in schizophrenic patients (-.692 < r < -.517,.003 < p <.030) but not in control subjects. Levels of all six synaptic mRNAs studied were increased in the younger (58-79 years) subgroup of schizophrenic patients compared to control subjects and older (80-95 years) subgroup of schizophrenic patients. CONCLUSIONS: That similar abnormalities were found for mRNAs encoding different synaptic vesicle and synaptic plasma membrane proteins suggests that they reflect overall neurodevelopmental abnormalities in synaptic connectivity in the temporal cortex of schizophrenic patients rather than changes in the number of synaptic vesicles per synapse or abnormalities in a specific synaptic function.

Abnormalities in 5-HT2A receptor mRNA expression in frontal cortex of chronic elderly schizophrenics with varying histories of neuroleptic treatment.

Alterations in the 5-HT2A receptor gene expression in the prefrontal cortex have been suggested to play a role in the pathophysiology and pharmacotherapy of schizophrenia. This study measured mRNA encoding 5-HT2A receptor in the left superior frontal gyrus from chronic elderly schizophrenics (n = 21) with varying neuroleptic-free intervals before death (72 hr to more than 5 years), and normal drug-free elderly controls (n = 14). Levels of 5-HT2A mRNA in schizophrenics correlated significantly and inversely with neuroleptic-free interval before death (r = -0.77; P < 0.0001). In schizophrenics who had been receiving neuroleptic until time of death, levels of 5-HT2A mRNA were similar to controls or greater. In schizophrenics who had been free of neuroleptic for more than six months levels of 5-HT2A mRNA were significantly lower than in controls. These results confirm previous findings of decreased expression of the 5-HT2A receptor gene in the frontal cortex of some schizophrenics and suggest that regulation of this gene may be involved in the therapeutic actions of typical neuroleptics.

Lack of a phenotype in transgenic mice aberrantly expressing COL2A1 mRNA because of highly selective post-transcriptional down-regulation.

We reported previously that a 1.9-kb 5'-fragment from the human COL1A1 gene drove transcription of a promoterless human COL2A1 gene in tissues of transgenic mice that normally express the COL1A1 but not the COL2A1 gene. In the present study, we have established that the aberrant transcription of the COL2A1 gene did not produce any gross or microscopic phenotype, because the transcripts were not efficiently translated in cells that do not normally express the COL2A1 gene. In two lines of transgenic mice, the mRNA levels from the transgene were 30% to 45% of the mRNA for the proalpha1(I) chain of type I procollagen, the most abundant mRNA in the same tissues. Analysis of collagens extracted from skin of the transgenic mice indicated that triple-helical type II collagen, with the normal pattern of cyanogen bromide peptides, was synthesized from the transgene. However, the level of type II collagen in skin was less than 2% of the level of type I collagen. Hybridization in situ indicated the presence of mRNA for both COL2A1 and COL1A1 in the same cells. Immunofluorescence staining for type II collagen, however, was negative in the same tissues. The results, therefore, indicated that many mesenchymal cells in the transgenic mice had high steady-state levels of the homologous mRNAs for type I and type II procollagen, but only the mRNAs for type I procollagen were efficiently translated.

Expression of NMDAR1, GluR1, GluR7, and KA1 glutamate receptor mRNAs is decreased in frontal cortex of "neuroleptic-free" schizophrenics: evidence on reversible up-regulation by typical neuroleptics.

Schizophrenics exhibit abnormalities in many memory-associated functions mediated by the frontal cortex. Glutamate receptors play key roles in learning and memory. Hence, abnormalities in glutamate receptors within the frontal cortex may be associated with schizophrenia. In addition, emerging evidence indicates that glutamate receptors may be involved in the actions of antipsychotic drugs. To test these hypotheses, we measured mRNAs encoding the NMDAR1, GluR1, GluR7, and KA1 subunits of glutamate receptor in the left superior frontal gyrus from 21 elderly schizophrenics with varying histories of antipsychotic drug treatment and nine normal drug-free elderly controls. There were significant negative correlations between NMDAR1, GluR1, GluR7, and KA1 mRNA levels and time without neuroleptic medication before death in schizophrenics, indicating that levels of the glutamate receptor mRNAs decline rapidly after drug withdrawal. Further analysis revealed that in "neuroleptic-free" (>6 months) schizophrenics, levels of NMDAR1, GluR1, GluR7, and KA1 mRNAs were significantly lower than in controls. By contrast, in schizophrenics who were receiving neuroleptics until death, levels of NMDAR1, GluR1, GluR7, and KA1 mRNAs did not differ significantly from controls. These findings indicate that decreased levels of NMDAR1, GluR1, GluR7, and KA1 mRNAs may be present in the frontal cortex of some schizophrenics and that typical neuroleptics may reversibly increase levels of these mRNAs.

Age-related abnormalities in expression of mRNAs encoding synapsin 1A, synapsin 1B, and synaptophysin in the temporal cortex of schizophrenics.

Synaptic abnormalities have been implicated in schizophrenia. In order to investigate synaptic pathology in schizophrenia, we examined levels of mRNAs encoding synaptophysin, synapsin 1A and synapsin 1B in the left temporal cortex from schizophrenics (n = 24) and from normal control individuals with no history of psychiatric illness (n = 10). Levels of synaptic mRNAs in the left superior temporal and left middle temporal gyrus declined significantly with age in schizophrenics, but not in controls. Dividing the diagnostic groups according to age (below and above 75 years), the data revealed that in "young" schizophrenics (age <75 years) levels of the three synaptic mRNAs in the left superior and left middle temporal gyri were approximately two times higher than in the age-matched controls. In the "old" schizophrenics (age >75 years) the levels of synaptic mRNAs in temporal cortex did not differ from age-matched controls. These findings further support the hypothesis that developmental synaptic abnormalities may be involved in the pathophysiology of schizophrenia.

Abnormal expression of serotonin transporter mRNA in the frontal and temporal cortex of schizophrenics.

Postmortem brain tissue was used to measure expression of serotonin transporter mRNA in the left frontal and left temporal cortex from schizophrenics (n = 24) and from normal control individuals with no history of psychiatric illness (n = 10). There was an approximately four-fold increase of serotonin transporter mRNA in Brodmann's area 9 and a two-fold decrease in Brodmann's areas 21 and 22. The changes in serotonin transporter mRNA were more prominent in schizophrenics who had received neuroleptic drugs within 1 week prior to death than in schizophrenics who were neuroleptic-free for more than 2 weeks prior to death, suggesting that the changes may be related to neuroleptic treatment. There was no correlation between serotonin transporter mRNA levels and the sex or age of schizophrenic and control cases or the postmortem delay intervals. These results support the hypothesis that region-specific changes in serotonin transporter expression occur in brains of some schizophrenics. Future studies are necessary to determine whether the changes in serotonin transporter expression are drug-related or are associated with the illness itself.

Identification of COL2A1 gene mutations in patients with chondrodysplasias and familial osteoarthritis.

OBJECTIVE: To use a recently developed procedure for analysis of blood leukocyte DNA to detect mutations in the gene for type II procollagen (COL2A1) in patients with cartilage diseases ranging from early-onset familial osteoarthritis (OA) to lethal chondrodysplasias. METHODS: The technique of denaturing gradient gel electrophoresis was used to scan polymerase chain reaction (PCR) products from 45 exons and exon-flanking sequences of the COL2A1 gene in more than 70 patients with cartilage diseases whose severity ranged from mild to lethal. PCR products with abnormal migrations were then sequenced. RESULTS: Among the 3 patients with lethal hypochondrogenesis who were analyzed, all 3 were found to have a mutation in the COL2A1 gene. Among 17 patients with spondyloepiphyseal or spondyloepimetaphyseal dysplasia, 2 well-defined and 2 probable mutations were found. Among 15 patients with the Wagner-Stickler syndrome, 2 well-defined and 2 probable mutations were found. Among 45 patients with early-onset familial OA, 1 probable mutation was found. CONCLUSION: Using the procedure developed for analysis of the COL2A1 gene, mutations were detected in > 20% of patients with chondrodysplasias and up to 2% of patients with early-onset familial OA. However, these percentages are only minimal estimates because all possible mutations in the gene cannot be detected with this procedure.

Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.

Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.

Tissue-specific expression of the gene for type I procollagen (COL1A1) in transgenic mice. Only 476 base pairs of the promoter are required if collagen genes are used as reporters.

Inconsistent data have been reported on the size of the promoter that is necessary for high levels of tissue-specific expression of the COL1A1 gene for type I procollagen. Some of the inconsistencies may be traced to the use of reporter gene constructs. Therefore, we prepared transgenic mice with modifications of the intact gene engineered so that the level of expression of the transgene could be assayed both as mRNA and protein that were similar to the products from the endogenous COL1A1 gene. The results with a mini-COL1A1 gene lacking 41 internal exons and introns indicated that the first intron and 90% of the 3'-untranslated region were not essential for tissue-specific expression. In a hybrid COL1A1/COL2A1 construct, a 1.9-kilobase 5'-fragment from the COL1A1 gene that contained only 476 of the promoter was linked to a promoterless 29.5-kilobase fragment of the human COL2A1 gene for type II procollagen. The hybrid COL1A1/COL2A1 construct was expressed as both mRNA and protein in tissues that normally synthesize type I procollagen but not type II procollagen. Apparently, 476 base pairs of the promoter are sufficient to drive tissue-specific expression of the COL1A1 gene and totally inappropriate expression of the COL2A1 gene.

A rapid and simple PCR-based method for isolation of cDNAs from differentially expressed genes.

Recently two techniques have been reported which use arbitrarily primed RT-PCR amplification of cDNA fragments from subsets of mRNAs to detect cDNA fragments from differentially expressed mRNAs. Here we report a simple and rapid PCR-based protocol to both detect and isolate cDNA fragments of up to 3000 base pairs from differentially expressed genes in two easy steps. To generate cDNAs from most mRNAs, the first step consisted of reverse transcription using a fully degenerated 6-mer oligonucleotide as primer. The second step consisted of PCR amplification of internal regions of the cDNAs with two or three longer primers with arbitrary but defined sequences. DNA fragments were easily displayed by agarose gel electrophoresis and then excised for direct use in cloning, sequencing, and Northern blot analysis. By repeating the PCR amplification (second step) on the same cDNA templates (first step) ten times with different sets of primers, over 170 discrete cDNA fragments were obtained from a single tissue. By combining the two-step procedure with 3'-RNA-anchored cDNA extension, additional DNA fragments can be generated from the same mRNA. The new procedure was used here to define 3600 bp of a new brain-specific mRNA.

Phenotypic variability and incomplete penetrance of spontaneous fractures in an inbred strain of transgenic mice expressing a mutated collagen gene (COL1A1).

Phenotype variability and incomplete penetrance are frequently observed in human monogenic diseases such as osteogenesis imperfecta. Here an inbred strain of transgenic mice expressing an internally deleted gene for the pro alpha 1(I) chain of type I procollagen (COL1A1) was bred to wild type mice of the same strain so that the inheritance of a fracture phenotype could be examined in a homogeneous genetic background. To minimize the effects of environmental factors, the phenotype was evaluated in embryos that were removed from impregnated females 1 d before term. Examination of stained skeletons from 51 transgenic embryos from 11 separate litters demonstrated that approximately 22% had a severe phenotype with extensive fractures of both long bones and ribs, approximately 51% had a mild phenotype with fractures of ribs only, and approximately 27% had no fractures. The ratio of steady-state levels of the mRNA from the transgene to the level of mRNA from the endogenous gene was the same in all transgenic embryos. The results demonstrated that the phenotypic variability and incomplete penetrance were not explained by variations in genetic background or levels in gene expression. Instead, they suggested that phenotypic variation is an inherent feature of expression of a mutated collagen gene.

Regulation of the alpha 1(I) collagen promoter in vascular smooth muscle cells. Comparison with other alpha 1(I) collagen-producing cells in transgenic animals and cultured cells.

We have previously reported that the expression of the ColCAT3.6 transgene containing 3.5 kilobases (kb) of alpha 1(I) collagen (COL1A1) promoter sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene paralleled the expression of the endogenous gene in several connective tissues. We report here that the activity of the reporter gene in aorta from 7-day-old transgenic mice is 10-64-fold lower than in tendon or bone, whereas the endogenous gene is highly expressed in all three tissues. In contrast, the COL1A1 minigene containing 2.3 kb of upstream sequence, the first five exon/intron units, the last six exon/intron units, and 2 kb of 3'-flanking sequence showed high CAT activity in aorta. These results suggest that cis sequences found in ColCAT3.6 mediate high levels of COL1A1 expression in bone and tendon, but not in vascular smooth muscle cells (VSMC), whereas sequences located within the minigene, but not found in ColCAT3.6, mediate VSMC-specific expression. Analysis of promoter activity in cultured cells derived from transgenic tissues further suggests the presence of VSMC-specific regulatory domains. Transient transfection studies, however, failed to shows differential regulation. These differences stress the importance of not relying exclusively on transient transfection data when mapping tissue-specific regulatory domains.

A single base mutation in the type II procollagen gene (COL2A1) that converts glycine alpha 1-247 to serine in a family with late-onset spondyloepiphyseal dysplasia.

A search for mutations in the gene for type II procollagen (COL2A1) was carried out in a family with late-onset spondyloepiphyseal dysplasia resulting in short sature, restricted mobility and severe pain in joints, deforming arthritis in the hips, and claudication. Analysis of the HindIII and VNTR polymorphisms at the COL2A1 gene in the family raised the possibility that the gene cosegregated with the disease. Screening for mutations in the COL2A1 gene using PCR-denaturing gradient get electrophoresis suggested a sequence variation in exon 19 of one allele of the COL2A1 gene in the proband. Direct sequencing of the PCR products for exon 19 revealed a single base mutation that converted the codon of -GGT- for glycine at alpha 1-247 to -AGT-, a codon for serine. The mutant that converted the present in all affected family members, but absent in nonaffected members and in a group of 50 unrelated healthy individuals. It was also absent in 20 unrelated patients with chondrodysplasia and 30 unrelated patients with early-onset osteoarthritis.

Tissue- and development-specific expression in transgenic mice of a type I procollagen (COL1A1) minigene construct with 2.3 kb of the promoter region and 2 kb of the 3'-flanking region. Specificity is independent of the putative regulatory sequences in the first intron.

Previous reports have provided inconsistent data as to the cis-regulatory elements that are essential for correct expression of the gene for the pro alpha 1 (I) chain of type I procollagen (COL1A1) in the many tissues in which the protein is synthesized. Here, two internally deleted minigene versions of the human COL1A1 gene were used to prepare transgenic mice. The constructs made it possible to test regulatory sequences in the normal context of the gene. Also, in contrast to the reporter genes used in previous experiments, the constructs made it possible to assay quantitatively expression of the exogenous genes relative to expression of the endogenous COL1A1 gene, both as mRNA and as protein. The average level of expression of the minigenes varied among three transgenic lines, but the ratio of expression of the minigenes to expression of the endogenous gene was the same in all transgenic mice of a given line. Within the same line, the ratio of expression was essentially the same in nine or more tissues in which expression of the endogenous gene varied widely. Also, the ratio of expression within a given line was the same in 15-day-old embryos and in mice ranging in age from 4 days to 4 months. In addition, the ratio remained constant during repair of a surgical wound. The results demonstrated, therefore, that the minigene constructs with about 2.3 kb of the promoter region and about 2 kb of the 3'-flanking region contained all of the sequences necessary for correct expression of the genes in a tissue-specific and development-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of an antisense oligonucleotide to inhibit expression of a mutated human procollagen gene (COL1A1) in transfected mouse 3T3 cells.

A series of antisense oligonucleotides were developed to inhibit specifically expression of a mutated exogenous gene for collagen without inhibiting expression of an endogenous gene for the same protein. The test system consisted of mouse NIH 3T3 cells that were stably transfected with an internally deleted construct of the human gene for the pro alpha 1(I) chain of type I procollagen [Olsen et al. (1991) J. Biol. Chem. 266, 1117]. The target site was a region at the 3' end of exon 1 and the first few nucleotides of intron 1 of the exogenous human gene that differed in sequence by nine nucleotides from the sequence of the endogenous mouse gene. Expression of the two genes was assayed by Western blot with cross-reacting antibodies and by steady-state levels of mRNAs. None of the oligonucleotides were effective in concentrations up to 25 microM when administered without any carrier. However, when administered with 5 or 10 micrograms/mL lipofectin, one of the oligonucleotides in concentrations of 0.1-0.2 microM inhibited expression of the exogenous gene from 50% to 80% without significant inhibition of expression of the endogenous gene. Also, a missense version of the same oligonucleotide had no significant effect, and the inhibition observed with the most effective oligonucleotide was abolished by a single base change. Time course experiments indicated that, after a 4-h treatment, inhibition appeared at 8 h and persisted for at least 22 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered ratio of collagen chains in bone of a patient with non-lethal osteogenesis imperfecta.

Bone from a patient with osteogenesis imperfecta contained type III collagen which was absent in control bone. The ratio of alpha 1(I)/alpha 2(I) in type I collagen of patient's bone was increased (2.9 vs. 2.3 +/- 0.2 in controls) and the ratio of dimers beta 11/beta 12/beta 22 was altered due to the increased beta 22 content. No abnormality was observed in collagen from the patient's skin. The altered composition of collagen in bone, but the normal composition in skin suggests that the disease in the patient is due to impaired regulation of the synthesis of collagens in bone, rather than by a mutation in one of the two type I collagen genes. Unlike in skin, all the type III collagen in patient's bone was pepsin-soluble indicating an inability of the bone to incorporate type III collagen into mature highly cross-linked extracellular matrix.