GABAB-mediated rescue of altered excitatory-inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR-hypofunction.

Reduced N-methyl-D-aspartate-receptor (NMDAR) signaling has been associated with schizophrenia, autism and intellectual disability. NMDAR-hypofunction is thought to contribute to social, cognitive and gamma (30-80 Hz) oscillatory abnormalities, phenotypes common to these disorders. However, circuit-level mechanisms underlying such deficits remain unclear. This study investigated the relationship between gamma synchrony, excitatory-inhibitory (E/I) signaling, and behavioral phenotypes in NMDA-NR1(neo-/-) mice, which have constitutively reduced expression of the obligate NR1 subunit to model disrupted developmental NMDAR function. Constitutive NMDAR-hypofunction caused a loss of E/I balance, with an increase in intrinsic pyramidal cell excitability and a selective disruption of parvalbumin-expressing interneurons. Disrupted E/I coupling was associated with deficits in auditory-evoked gamma signal-to-noise ratio (SNR). Gamma-band abnormalities predicted deficits in spatial working memory and social preference, linking cellular changes in E/I signaling to target behaviors. The GABA(B)-receptor agonist baclofen improved E/I balance, gamma-SNR and broadly reversed behavioral deficits. These data demonstrate a clinically relevant, highly translatable neural-activity-based biomarker for preclinical screening and therapeutic development across a broad range of disorders that share common endophenotypes and disrupted NMDA-receptor signaling.

Assessment of NMDA receptor NR1 subunit hypofunction in mice as a model for schizophrenia.

N-methyl-D-aspartate receptors (NMDARs) play a pivotal role in excitatory neurotransmission, synaptic plasticity and brain development. Clinical and experimental evidence suggests a dysregulation of NMDAR function and glutamatergic pathways in the pathophysiology of schizophrenia. We evaluated electrophysiological and behavioral properties of NMDAR deficiency utilizing mice that express only 5-10% of the normal level of NMDAR NR1 subunit. Auditory and visual event related potentials yielded significantly increased amplitudes for the P20 and N40 components in NMDAR deficient (NR1(neo)-/-) mice suggesting decreased inhibitory tone. Compared to wild types, NR1(neo)-/- mice spent less time in social interactions and showed reduced nest building. NR1(neo)-/- mice displayed a preference for open arms of a zero maze and central zone of an open field, possibly reflecting decreased anxiety-related behavioral inhibition. However, locomotor activity did not differ between groups in either home cage environment or during behavioral testing. NR1(neo)-/- mice displayed hyperactivity only when placed in a large unfamiliar environment, suggesting that neither increased anxiety nor non-specific motor activation accounts for differential behavioral patterns. Data suggest that NMDAR NR1 deficiency causes disinhibition in sensory processing as well as reduced behavioral inhibition and impaired social interactions. The behavioral signature in NR1(neo)-/- mice supports the impact of impaired NMDAR function in a mouse model with possible relevance to negative symptoms in schizophrenia.

Messenger RNA editing of the human serotonin 5-HT2C receptor.

RNA encoding the rat serotonin 5-HT2C receptor undergoes editing whereby one to four adenosines are converted to inosines. This conversion can change up to three codons out of a stretch of five in the second intracellular loop of the receptor. RNA editing of the rat 5-HT2C receptor that changes all three codons was shown previously to alter intracellular signaling by 5-HT without changing its receptor-binding affinity. We analyzed 5-HT2C receptor editing in human brain and hypothalamic RNA samples and confirmed that all four adenosine editing sites observed in rat were also present in human samples. Additionally, we identified a novel editing site in the middle edited codon that extends the repertoire of 5-HT2C receptors by six additional protein isoforms. We observed that editing reduces both the binding affinity and functional potency of agonists for recombinant human 5-HT2C receptor isoforms. This effect on binding affinity was proportional to the agonist's intrinsic activity, with full agonists most affected, and antagonists showing no effect. These data suggest that RNA editing may alter coupling energetics within the ternary complex, thereby altering agonist binding affinities, G protein coupling, and functional responses. RNA editing may thus provide a novel mechanism for regulating 5-HT synaptic signaling and plasticity.

Cataractogenesis in transgenic mice containing the HIV-1 protease linked to the lens alpha A-crystallin promoter.

Several lines of transgenic mice were generated with either active or inactive forms of the human immunodeficiency virus type 1 (HIV-1) protease gene under the control of the mouse lens alpha A-crystallin promoter. Mice bearing the inactive protease coding sequence displayed no gross abnormalities in the lens, while mice with the active protease developed time-dependent bilateral cataracts. One line, TG61, developed cataracts in utero while the second line, TG72, developed cataracts postnatally. TG61 mice, homozygous for the transgene, developed severe microphthalmia and were significantly smaller than the control mice at postnatal day 30. two-dimensional-polyacrylamide gel electrophoresis analysis of the protein profiles of TG72 and TG61 lenses revealed extensive modifications in the lens crystallins. Proteolysis in the homozygous TG72 mouse lenses began at postnatal day 20 with the disappearance or partial loss of beta B1-, beta B3-, and beta A3-crystallins and the appearance of crystallin fragments. Protein leakage and the gradual breakdown of cytoskeletal elements also occurred. In contrast, the opacification of the homozygous TG61 lenses appeared to have been influenced by differentiation and developmental processes. It appears that HIV-1 protease expression activates other proteases, and these enzymes, in concert with HIV-1 protease, are responsible for the protein modifications that eventually result in the opacification of the lens.

Sequence requirements for synthetic peptide-mediated translocation to the nucleus.

The abilities of 18 synthetic peptides to target a carrier protein to the nucleus following microinjection into the cytoplasm of HeLa cells were determined. Eight of the sequences chosen for synthesis were based on published nuclear targeting regions as determined by gene fusion and deletion experiments. Six of these sequences were found to be effective when mimicked by a synthetic peptide and conjugated to a carrier protein. One additional peptide was based on a region of lamin L1, a nuclear protein from Xenopus laevis, in which the nuclear targeting region had not been previously investigated. This peptide was also able to target a carrier protein to the nucleus. Eight other peptides which resemble the known targeting signals had little or no nuclear targeting ability. Peptides which were able to target a carrier protein to the nucleus did so within 45 min of injection into the cytoplasm. Two peptides with little or no apparent nuclear targeting ability after 45 min were examined for longer times as well. No increase in nuclear accumulation was observed between 45 min and 4 h after cytoplasmic injection. Comparison of the sequences which were effective at nuclear targeting with those that were not revealed a possible consensus sequence for peptide-mediated nuclear transport.

Chemical synthesis and expression of a gene encoding HIV-1 TAT protein.

A gene encoding the human immunodeficiency virus-1 (HIV-1) TAT protein was chemically synthesized and expressed in HeLa cells and in a cell-free system. To facilitate both the assembly of the synthetic gene and further mutagenesis and gene fusion studies, several unique restriction endonuclease cleavage sites were included in the coding sequence without altering the encoded protein sequence. The synthetic TAT coding sequence was fused to a translation start signal and placed under SV40 early transcriptional control. Co-transfection of the TAT-encoding synthetic gene together with a reporter gene (chloramphenical acetyl transferase or beta-galactosidase) linked to an HIV LTR confirmed that the synthetic gene product exhibits similar activity to TAT expressed from HIV genomic DNA in the transactivation of the LTR. TAT mRNA prepared by cell-free transcription of the synthetic TAT coding sequence was also shown to produce functional TAT following microinjection into HeLa-derived cells containing an integrated reporter gene with the HIV LTR linked to beta-galactosidase.

Regulation of c-myc RNA and its proteins in Daudi cells by interferon-beta.

It has been shown previously that interferons (IFNs) -alpha and -beta cause a reduction in the steady-state level of poly(A) c-myc RNA in the Burkitt lymphoma, Daudi. In this report we show that the c-myc RNA reduction is not mediated by simple changes in the poly-adenylation of either nascent or existing c-myc transcripts, since similar reductions of c-myc sequences were observed in poly(A) and total cellular RNA preparations from IFN-beta-treated cells. Furthermore, the first exon of c-myc RNA in Daudi cells contains several mutations, suggesting that the germ line configuration of the first exon is not essential for the IFN-beta-mediated regulation. The c-myc RNA reduction was also detected in cells whose protein synthesis was inhibited by more than 95% with cycloheximide or emetine. We surmise that neither sustained nor IFN-induced protein synthesis is required for the c-myc RNA regulation. Antisera raised against either the carboxy- or amino-terminal c-myc peptides precipitate in Daudi cells proteins of 66,000 and 63,000 daltons. In cells treated with IFN-beta, the amounts of these proteins are reduced by 46-74% which is in agreement with the reduction detected at the level of c-myc RNA.

Nonidentical induction of the guanylate binding protein and the 56K protein by type I and type II interferons.

Upon the addition of interferon (IFN) to cultured human cells, the expression of genes encoding the 56K and the guanylate binding protein (GBP) is specifically induced. We have analyzed their expression at the protein and the mRNA levels and studied how their regulation differs in cells treated with different IFNs. In the type I IFN (alpha and beta)-treated cells, we detected the accumulation of the 56K protein primarily in the cytoplasm. The 56K protein was undetectable in untreated cells or in cells treated with type II IFN (IFN-gamma). In contrast, a greater amount of GBP was synthesized in cells treated with type II IFN than in cells treated with type I IFN. The differential induction of these two proteins correlates well with the relative amounts of their mRNAs in type I and type II IFN-treated cells. In addition, the IFN-induced synthesis of the 56K protein was found in certain cell lines in which the GBP synthesis was not detected. These results suggest that the regulation of these two genes requires dissimilar factors which are activated or induced to different extents by type I and type II IFNs.

Interferon regulates c-myc gene expression in Daudi cells at the post-transcriptional level.

c-myc gene mRNA is reduced by greater than 75% in the human lymphoblastoid cell line Daudi when growth is inhibited by treatment with human interferon beta (IFN-beta). In the present communication, we describe the effect of IFN-beta treatment on transcription of the c-myc gene and on the steady-state level of c-myc mRNA in the cytoplasm of Daudi cells. The results show that, although the rate of c-myc transcription is not significantly different in nuclei isolated either from untreated cells or from those treated with IFN-beta for 3 or 24 hr, the level of c-myc mRNA in the cytoplasm is reduced by 60% within 3 hr of IFN-beta treatment. These results suggest that IFN-beta regulates the c-myc mRNA at a post-transcriptional level. These results are in contrast to the regulation of two IFN-beta-induced genes that under identical conditions are regulated in these cells at the transcriptional level. We have also detected induction of the (2'-5')oligoadenylate synthetase (2-5A synthetase) gene in IFN-beta-treated Daudi cells. Since certain c-myc transcripts have the capacity to form double-stranded RNA regions, we propose that one mechanism by which c-myc could be regulated post-transcriptionally in IFN-beta-treated cells is by activating, through its own double-strandedness, the 2-5A synthetase/RNase L endonuclease system, which would cause selective degradation of the c-myc RNA.

Interferon-induced proteins. Purification and characterization of a 15,000-dalton protein from human and bovine cells induced by interferon.

Human interferons induce a protein of 15,000 daltons in human and bovine cells. This protein is located in the cytoplasm in a soluble form and is induced by concentrations of interferon which induce the antiviral state. Messenger RNA prepared from interferon-treated human and bovine cells contains a mRNA which yields on translation in vitro a protein similar in size to the 15-kDa protein induced by interferon in vivo. The human protein has been purified to homogeneity from interferon-treated human cells by ion-exchange chromatography and reverse-phase high-performance liquid chromatography. A comparison of the peptides generated by V8 protease from the human and bovine 15-kDa proteins reveals that the two proteins are similar but not identical.

Transcriptional induction of two genes in human cells by beta interferon.

The binding of interferons to distinct cell surface receptors leads to the induction of synthesis of several unique polypeptides and their corresponding mRNAs (1-6). We have isolated two cDNAs that are complementary to nuclear RNA whose synthesis is induced from undetectable levels to maximal rates of transcription within 30-60 min after the addition of beta interferon to human fibroblasts or to HeLa cells. These results prove that a single polypeptide can, by binding to a specific plasma membrane receptor, promptly activate the transcription of a defined set of genes.

Selective reduction of c-myc mRNA in Daudi cells by human beta interferon.

Under normal growth conditions, the human lymphoblastoid cell line Daudi expresses high levels of c-myc mRNA. These cells are also sensitive to growth inhibition by interferons. We have compared the levels of mRNA for the c-myc in untreated and human beta interferon (IFN-beta)-treated Daudi cells by RNA dot-blot and blot-hybridization analysis methods. Using a synthetic oligonucleotide complementary to the human c-myc mRNA as the probe, we detected a more than 75% reduction in the c-myc hybridizable poly(A)+ RNA in the IFN-beta-treated cells. This reduction in the c-myc mRNA appears to be selective because the level of actin mRNA is not significantly affected by the IFN-beta treatment. In addition, neither in vitro translation of mRNA extracted from IFN-beta-treated cells nor in vivo synthesis of cellular proteins in IFN-beta-treated cells are quantitatively affected. We surmise that the selective reduction in the amount of c-myc mRNA in IFN-beta-treated Daudi cells may be related to the IFN-induced inhibition of the Daudi tumor cell growth.

Microinjected pBR322 stimulates cellular DNA synthesis in Swiss 3T3 cells.

When pBR322 is manually microinjected into the nuclei of quiescent Swiss 3T3 cells it stimulates the incorporation of [3H]thymidine into DNA. The evidence clearly shows that this increased incorporation that is detected by in situ autoradiography in microinjected cells represents cellular DNA synthesis and not DNA repair or plasmid replication. The effect is due to pBR322 and not due to impurities, mechanical perturbances due to the microinjection technique, or aspecific effects. This stimulation is striking in Swiss 3T3 cells. Some NIH 3T3 cells show a slight stimulation, but hamster cells, derived from baby hamster kidney (BHK) cells, are not stimulated when microinjected with pBR322. The preliminary evidence seems to indicate that the integrity of the pBR322 genome is important for the stimulation of cellular DNA synthesis in quiescent Swiss 3T3 cells. These results, although of a preliminary nature, are of interest because they indicate that a prokaryotic genome may alter the cell cycle of mammalian cells. From a practical point of view the stimulatory effect of microinjected pBR322 on cellular DNA synthesis has a more immediate interest, because pBR322 is the vector most commonly used for molecular cloning and 3T3 cells are very frequently used for gene transfer experiments.

Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene.

The biological activity of several deletion mutants of simian virus 40, cloned in pBR322, was determined. Three functions of the simian virus 40 A gene were studied: (i) the ability to express T antigen; (ii) the ability to induce cell DNA replication; and (iii) the ability to reactivate silent rRNA genes in hybrid cells. Recombinant plasmid DNA was introduced into cells by manual microinjection or by transfection. The results (together with previous reports) indicate that the critical sequences for these three functions are located separately on the simian virus 40 A gene, as follows: (i) the sequences necessary for the detection of the common antigenic determinant of T antigen extend from nucleotide 4147 to nucleotide 4001 (map units 0.45 to 0.42); (ii) the sequences critical for the stimulation of cell DNA synthesis extend from nucleotide 4327 to nucleotide 4001 (map units 0.49 to 0.42); and (iii) those critical for the reactivation of rRNA genes extend approximately from nucleotide 3827 to nucleotide 3526 (map units 0.39 to 0.33).

Microinjection of cloned SV40 DNA fragments in the study of cell proliferation.

DNA recombinant technology and the manual microinjection technique were used to study the base sequences in the SV40 early gene coding for cell DNA replication and nucleolar activation. Sequences critical for rRNA gene activation are located between nucleotides 3826 to 3506 (0.39-0.33 m.u.). Base sequences from nucleotide 4350 to 4190 (0.49-0.46 m.u.) are required for cellular DNA replication. Major T antigen determinant is coded by a sequence extending from nucleotide 4190 to 3880 (0.46-0.42 m.u.). Considering that an increase in cell size is regularly accompanied by an increase in the cellular amount of rRNA, nucleolar genes should be a target for growth-in-size signals. Therefore, the SV40 early gene presents a domain responsible for cell growth in size, and another separate but proximate region coding for cellular DNA replication.

Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks.

Woodchuck hepatitis virus (WHV), like the related hepatitis B virus, induces in its natural host hepatocellular carcinomas that contain integrated viral sequences. As a first step in determining whether and how the integrated sequences contribute to formation of the tumors in which they are found, we have cloned two such integrations of WHV and have determined their structure by restriction mapping and heteroduplex electron microscopy. The identity of the cloned sequences was confirmed by comparison of restriction sites in the clones with those located by Southern blot analysis of tumor DNA. Viral sequences in both integrations are extensively rearranged, and in neither were all parts of the viral genome represented. In this respect, the behavior of WHV in vivo is similar to that of other DNA tumor viruses that have been studied in vitro. We discuss the implications of these results in relation to possible mechanisms for tumor induction by WHV.

Characterization and biological activity of cloned simian virus 40 DNA fragments.

The biological activity of fragments of the SV40 genome was determined by manual microinjection of the fragments into the nuclei of mammalian cells. Fragments of the SV40 A gene (that codes for the T antigens) were obtained either directly by digestion with restriction endonucleases or after cloning into plasmid pBR322. Three different biological activities were studied: expression of T antigen, induction of cell DNA synthesis, and, in a few cases, reactivation of repressed ribosomal RNA genes. By using a number of fragments with deletions in the various portions of the SV40 A gene, we have been able to conclude that: 1) the sequences from 0.65 to 0.51 map units are not needed for the induction of cell DNA synthesis; 2) the sequences from 0.42 to 0.17 map units are not needed for the induction of cell DNA synthesis; and 3) the critical sequences for the induction of cell DNA synthesis, 0.51 to 0.42 map units, are different from those necessary for the reactivation of repressed ribosomal RNA genes (0.39-0.33 map units). These results indicate that the information for these two fundamental processes of cell proliferation resides in two separate and distinct domains of the SV40 A gene.

Identification of adenovirus 2 early genes required for induction of cellular DNA synthesis in resting hamster cells.

tsAF8 cells are temperature-sensitive (ts) mutants of BHK-21 cells that arrest at the nonpermissive temperature in the G1 phase of the cell cycle. When made quiescent by serum restriction, they can be stimulated to enter the S phase by 10% serum at 34 degrees C, but not at 40.6 degrees C. Infection by adenovirus type 2 or type 5 stimulates cellular DNA synthesis in tsAF8 cells at both 34 and 40.6 degrees C. Infection of these cells with deletion Ad5dl312, Ad5dl313, Ad2 delta p305, and Ad2+D1) and temperature-sensitive (H5ts125, H5ts36) mutants of adenovirus indicates that the expression of both early regions 1A and 2 is needed to induce quiescent tsAF8 cells to enter the S phase at the permissive temperature. This finding has been confirmed by microinjection of selected adenovirus DNA fragments into the nucleus of tsAF8 cells. In addition, we have shown that additional viral functions encoded by early regions 1B and 5 are required for the induction of cellular DNA synthesis at the nonpermissive temperature.