Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys.

Apoptosis is a morphologically defined form of programmed cell death seen in a variety of circumstances, including immune cell selection, carcinogenesis and development. Apoptosis has very recently been seen after ischemic or traumatic injury to the central nervous system (CNS), suggesting that active cell death as well as passive necrosis may mediate damage after CNS injury. After spinal cord injury (SCI) in the rat, typical post-traumatic necrosis occurred, but in addition, apoptotic cells were found from 6 hours to 3 weeks after injury, especially in the spinal white matter. Apoptotic cells were positive for oligodendrocyte markers. After SCI in monkeys, apoptotic cells were found within remote degenerating fiber tracts. Both secondary degeneration at the site of SCI and the chronic demyelination of tracts away from the injury appear to be due in part to apoptosis. As cytokines have been shown to mediate oligodendrocyte death in vitro, it seems likely that chronic demyelination after CNS injury shares features with chronic degenerative disorders like multiple sclerosis.

Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer's disease and in response to experimental lesions in rat.

A hippocampal poly(A) RNA, pADHC-9, was cloned by differential screening of a human hippocampal cDNA library. By RNA blot analysis, pADHC-9 was elevated 2-fold in Alzheimer's disease hippocampus. In situ analyses identified pADHC-9 expression in pyramidal and non-pyramidal cells of the hippocampus and entorhinal cortex. Nucleotide sequence analysis identified pADHC-9 as a potential human homolog of rat sulfated glycoprotein 2 (SGP-2). SGP-2 expression increased in rat hippocampus following experimental lesions that mimic intrinsic neuronal loss and/or deafferentation. The function of pADHC-9 in brain has not been defined, but in serum, a similar protein inhibits complement-dependent cytolysis. Increased expression of pADHC-9 in Alzheimer's disease hippocampus may be a compensatory response mounted to retard a complement-driven neurodegenerative cascade.

Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site.

The 5' ends of dihydrofolate reductase (DHFR)-specific transcripts have been mapped in the 5'-flanking region of the amplified DHFR gene of the human methotrexate-resistant cell line 6A3 by primer extension and S1 protection experiments. The main 5' end, at position -71 relative to the first nucleotide of the DHFR reading frame, corresponds to the recently identified main transcription initiation site for the DHFR gene and pertains to transcripts representing approximately 99% of the DHFR-specific polysomal polyadenylic acid-containing RNA, and including the previously described DHFR mRNAs with sizes of 3.8, 1.0, and 0.8 kilobases. At least six other minor 5' ends have been mapped to nucleotide positions -449 to -480 upstream of the DHFR gene and pertain to approximately 1% of the DHFR-specific polysomal polyadenylic acid-containing RNA. These upstream initiating transcripts appear to include five major discrete species with sizes of 4.3, 3.8, 3.1, 2.1, and 1.0 kilobases and four minor ones with sizes of 7.3, 5.0, 1.4, and 0.8 kilobases. These species, with the exception of those of 3.1- and 2.1-kilobase sizes, also have been found in VA2-B cells, the parental line of 6A3, and in HeLa cells. The upstream initiating transcripts present in all three cell lines are increased in amount in 6A3 cells as compared with the other cell lines, in about the same proportion as the three identified DHFR mRNAs.

Human dihydrofolate reductase gene organization. Extensive conservation of the G + C-rich 5' non-coding sequence and strong intron size divergence from homologous mammalian genes.

The complete human dihydrofolate reductase (DHFR) gene has been cloned from four recombinant lambda libraries constructed with the DNA from a methotrexate-resistant human cell line with amplified DHFR genes. The detailed organization of the gene has been determined by restriction mapping of the cloned fragments and DNA sequencing of all the protein coding regions and adjacent intron segments, and shown to correspond to that of the native human DHFR gene. The gene spans a length of approximately 29 X 10(3) bases from the ATG initiator codon to the end of the 3' untranslated region, and contains five introns that interrupt the protein coding sequence. The number and positions of introns are identical to those found in the mouse gene. By contrast, the size of the homologous introns (with the exception of the first one) varies greatly, up to several fold, in the genes from man, mouse and Chinese hamster; the intron sequences also exhibit a great divergence, except in the junction regions. A striking sequence homology, extending over several hundred nucleotides, exists between the human and mouse gene 5' non-coding regions. These regions are characterized by an unusually high G + C content, 72% and 66% in the human and mouse genes, respectively, which is maintained in the first coding segment and first intron, and is in sharp contrast to the relatively low G + C content (approximately 40%) of the remainder of the gene.

A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA.

The presence of dihydrofolate reductase (DHFRase)-specific sequences that, in contrast to the normal DHFRase gene, are not amplified in a methotrexate-resistant cell line, has been detected in the DNA from human sperm and from several human cell lines. DNA fragments containing some of these sequences have been isolated from a cosmid library of human sperm DNA. One of these fragments contains a DHFRase pseudogene (psi HD1) that completely lacks introns, has 92% sequence homology to the corresponding region of normal DHFRase complementary DNA, but exhibits several alterations that make it nonfunctional. The sequence analysis of the inserts of four different plasmids containing the reading frame and varying lengths of the 3' non-coding regions of human DHFRase-specific cDNAs has revealed that the 3' non-coding segments all are colinear in their corresponding portions. Furthermore, the data indicate that the cDNA of one of the plasmids is probably derived from the smallest of the three main human DHFRase messenger RNAs, the 0.8 X 10(3) base (0.8 kb) mRNA, the cDNA of two others, from the 1.0 kb mRNA, and the cDNA of the fourth, from a longer mRNA. These results are consistent with the idea that the multiple forms of DHFRase mRNA in human cells derive from the same gene by different transcription or RNA-processing events. Moreover, the sequence comparison between the psi HD1 and the different DHFRase cDNAs clearly indicates that, if an mRNA intermediate has participated in the formation of this pseudogene, a form of mRNA larger than the 1.0 kb mRNA, probably the 3.8 kb mRNA, must have been involved.

Rapid corticosterone-induced changes in gene expression in rat hippocampus display type II glucocorticoid receptor specificity.

Corticosteroids influence a wide range of neuronal activities by binding to either of two different glucocorticoid receptors found in rat brain. To investigate genomic responses in brain to stress levels of circulating corticosterone (CORT), we isolated hippocampal total RNA and poly(A)-containing RNA from rats treated with 10 mg/day CORT or vehicle. RNA translation products were resolved by 2-dimensional gel electrophoresis and fluorography. Select changes in four translation products after acute CORT treatment were inferred from up to 100-fold increases in three polypeptides and a 2-fold decrease in another. While adrenalectomy decreased levels of the inducible RNA sequences (adrenalectomized vs. intact controls), CORT increased the inducible sequences above their levels in intact controls. Rapid increases within 2 h of CORT treatment were seen for RNAs coding for 35, 33, and 20 kilodalton polypeptides. However, RNA coding for a 50 kilodalton polypeptide had a delayed decrease, first seen after 32 h CORT. The CORT increases displayed type II glucocorticoid receptor-specificity: RU 28362 greater than or equal to CORT greater than aldosterone greater than dihydrotestosterone = control. Since type II receptors are only substantially occupied by stress levels of CORT, these changes in gene expression are candidates for molecular stress responses in the brain.

Glucocorticoid endangerment of hippocampal neurons does not involve deoxyribonucleic acid cleavage.

Glucocorticoids (GCs) are highly pathogenic if secreted in excess. Recent work shows that such deleterious consequences include damage to the hippocampus, a principal neural target site for GCs. Excessive chronic exposure to GCs accelerates senescent hippocampal neuron loss, while the presence of GCs at the time of neurological insults, such as seizure or hypoxia-ischemia, exacerbates hippocampal damage. The present study determines whether GCs endanger hippocampal neurons through the same mechanism by which they damage lymphocytes. GC-induced lymphocytolysis involves cleavage of chromosomal DNA, most likely through steroid induction of a nuclease that produces a characteristic ladder of fragmented DNA. Moreover, inhibition of DNA repair using the poly(ADP-ribose) synthetase inhibitor benzamide exacerbates GC-induced lymphocytolysis. We replicated this GC-induced fragmentation of DNA in thymocytes, but observed the absence of a similar fragmentation in DNA from primary hippocampal cultures under conditions in which GCs exacerbate the toxic effects of the excitotoxin kainic acid. Furthermore, under such conditions benzamide did not worsen the GC/kainic acid toxicity. These observations suggest that GCs endanger hippocampal neurons through a different mechanism, one that seems likely to be less sterotyped and simple than this cascade of apoptosis in lymphocytes.

The nucleotide sequence of the cDNA coding for the human dihydrofolic acid reductase.

The nucleotide sequence of the human dihydrofolic acid reductase (DHFR) reading frame has been derived from the analysis of human DHFR cDNA. This sequence and the corresponding amino acid sequence have been compared with those available for the enzyme and its coding segment from other organisms. There is an 89% nucleotide sequence homology between the human DHFR reading frame and the mouse coding sequence. Furthermore, amino acid-sequence homologies of 74%, 81% and 89% has been found between human DHFR and chicken, bovine and mouse DHFR, respectively.

Molecular modeling and pharmacological analysis of species-related histamine H(3) receptor heterogeneity.

Presynaptic histamine H(3) receptors (H(3)R) regulate neurotransmitter release in the central nervous system, suggesting an important role for H(3) ligands in human diseases such as cognitive disorders, sleep disturbances, epilepsy, or obesity. Drug development for many of these human diseases relies upon rodent-based models. Although there is significant sequence homology between the human and rat H(3)Rs, some compounds show distinct affinity profiles. To identify the amino acids responsible for these species disparities, various mutant receptors were generated and their pharmacology studied. The N-terminal portion was shown to determine the species differences in ligand binding since a chimeric H(3)R containing N-terminal human and C-terminal rat receptor sequences exhibited similar pharmacology to the human receptor. Sequence analysis and molecular modeling studies suggested key amino acids at positions 119 and 122 in transmembrane region 3 play important roles in ligand recognition. Mutant receptors changing amino acids 119 or 122 of the human receptor to those in the rat improved ligand binding affinities and functional potencies of antagonist ligands, confirming the significant role that these amino acids play in species-related pharmacological differences. A model has been developed to elucidate the ligand receptor interactions for H(3)Rs, and pharmacological aspects of this model are described.

Growth hormone regulation of hepatic glutamine synthetase mRNA levels in rats.

The expression of glutamine synthetase (GS) in the rat liver is dependent on pituitary growth hormone (GH). RNA blot hybridizations revealed that in hypophysectomized rats the level of glutamine synthetase mRNA was dramatically reduced in liver but not brain. This drop of GS mRNA in the liver results in a reduction of GS enzyme activity as well. Two other messages, phosphoenolpyruvate carboxykinase and glycerol phosphate dehydrogenase were not diminished in the liver, indicating that the effects of hypophysectomy on hepatic GS expression are specific and not part of a general reduction in transcription due to lack of pituitary factors. Daily administration of rat pituitary growth hormone caused an increase in the levels of hepatic GS mRNA as well as enzyme activity. In situ hybridization of normal liver sections with the GS antisense message showed an abundant amount of message confined to the region around each central vein of the hepatic acini, while in the hypophysectomized animal the message for GS is greatly reduced but still only located in hepatocytes surrounding the central vein. Hypophysectomized animals given GH replacement showed a substantial increase in the amount of exposed silver grains only around the central veins. This indicates that GH does not influence the cellular position of GS expression nor the viability of those hepatocytes that express the enzyme, but it does regulate the quantity of GS in the liver through changes in the levels of GS mRNA.

CR16, a novel proline-rich protein expressed in rat brain neurons, binds to SH3 domains and is a MAP kinase substrate.

CR16 is a glucocorticoid-regulated gene expressed in subpopulations of neurons in the brain, including the hippocampus. The CR16 open reading frame encodes a 45 kDa protein containing 32% proline. To begin characterizing the CR16 protein, a rabbit polyclonal antibody was raised against an Escherchia coli-produced fusion protein containing amino acids 370-438 of CR16. The antibody identifies a protein doublet of 68 and 72 kDa by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) from hippocampal extracts and from insect cells expressing the CR16 open reading frame from a baculovirus construct. However, when hippocampal extracts are electrophoresed on nondenaturing polyacrylamide gels, the CR16 protein migrates as a 48 kDa protein that better correlates with the size of the open reading frame. Examination of the primary amino acid sequence reveals at least 12 sequence homologies to the abl-SH3 binding domain consensus sequence XPXXPPP psi XP. In addition, CR16 has at least 36 copies of the PXXP motif, which is contained in all known SH3 binding domains. Solution and filter binding assays confirm that CR16 selectively binds SH3 domains. The CR16 primary amino acid sequence also contains at least eight consensus MAP kinase phosphorylation sites, five of which are in the potential SH3 binding domains. The CR16 protein, immunoprecipitated from rat brain, is an in vitro substrate for the purified enzyme. However, phosphorylation of CR16 does not greatly affect the binding of the various SH3 domains in our assay system. These data strongly suggest that the function of CR16 is to mediate one or more signal transduction pathways in CNS neurons, in addition to being a glucocorticoid-regulated gene.

Messenger RNA for glial fibrillary acidic protein is decreased in rat brain following acute and chronic corticosterone treatment.

RNA coding for a 50 kDa polypeptide decreased by 50% in 5 brain regions after corticosterone (CORT) treatment (40 mg/kg for 3 days). By hybrid selection and in vitro translation, the 50 kDa polypeptide is identified as glial fibrillary acidic protein (GFAP). Hippocampal GFAP mRNA (2.9 kb) decreases in a dose-dependent manner in response to CORT by RNA blot hybridization using a mouse GFAP cRNA probe; a similar decrease in response to the glucocorticoid agonist, RU 28362, is consistent with a type II glucocorticoid receptor-mediated effect. GFAP mRNA is decreased in both hippocampus and cortex following acute (1-3 days) and chronic (3 days to 3 months) CORT treatment. GFAP gene expression is disinhibited in the rat hippocampus by 7 days post adrenalectomy but not by 3 days. Finally, two clones (CR46 and CR59) that were isolated from a rat hippocampal cDNA library by differential hybridization, show decreased RNA abundance in CORT-treated rats compared to controls. A partial DNA sequence derived from the two clones exhibits 94% nucleotide identity and 96% derived amino acid identity with mouse GFAP mRNA. These results indicate that GFAP mRNA is under negative regulation by glucocorticoids and suggests that glucocorticoids may be used to inhibit GFAP gene expression in vivo in order to assess the role of GFAP in temporal aspects of central nervous system damage.

Effects of a specific glucocorticoid receptor antagonist on corticotropin releasing hormone gene expression in the paraventricular nucleus of the neonatal rat.

Mechanisms controlling the synthesis of corticotropin releasing hormone (CRH) in neonatal rats, and the ontogeny of glucocorticoid (GC) feedback control of hypothalamic CRH remain unknown. Specific issues are whether stress induces up-regulation of CRH gene expression during the first postnatal week, and the role of GC feedback, at the hypothalamic level, in the stress-hyporesponsive period. We studied the ontogeny of the negative feedback regulation of CRH gene expression by GC in the paraventricular nucleus (PVN). We implanted chronic cannulae containing a GC-receptor antagonist, RU 38486, in rats on postnatal days 3 to 13. Three days later, animals were sacrificed, and brains were analyzed for CRH-messenger RNA (CRH-mRNA), using semi-quantitative in situ hybridization. Animals implanted with cholesterol-containing cannulae served to evaluate the stressful effect of implantation on CRH-mRNA abundance. The presence of GC receptor messenger RNA (GR-mRNA) in the PVN of neonatal rats was also determined. RU 38486 did not increase CRH-mRNA abundance during the first postnatal week, despite the presence of GR-mRNA in the PVN. Chronic-implantation stress also failed to increase CRH synthesis. CRH gene expression in the PVN was enhanced in infant rats implanted with RU-38486 on postnatal day 9 or later. Cholesterol implantation on days 9, 10 (but not later), resulted in increased PVN-CRH-mRNA. Thus, CRH-mRNA is up-regulated by chronic blockade of GC receptors only subsequent to the eighth postnatal day. Furthermore, such blockade does not affect the response of CRH-mRNA to chronic stress in the neonatal rat.

Changes in gene expression in hippocampus in response to glucocorticoids and stress.

Glucocorticoid hormones, acting through two types of intracellular receptors to modulate gene activity, have diverse behavioral, neurochemical and neurodegenerative effects in hippocampus. We have previously cloned hippocampal mRNAs that respond to the endogenous glucocorticoid, corticosterone (CORT): glycerol phosphate dehydrogenase (EC 1.1.1.8; GPDH), an oligodendrocyte marker; CR16, whose sequence is not yet identified; and glial fibrillary acidic protein (GFAP), a marker of astroglial reactivity. In these studies, we have subjected rats to 2 hr vibratory stress as a treatment that raises circulating CORT levels, and analyzed changes in GPDH, CR16 and GFAP mRNAs in rat hippocampus. Only GPDH mRNA responded to stress in intact rats; GPDH mRNA did not respond to the same treatment in rats where the adrenal source of CORT had been removed surgically. The lack of stress responsiveness of CR16 and GFAP mRNAs, despite elevated corticosterone levels, is consistent with their slower (greater than 2 hr but less than 8 hr) response to administered CORT. These studies indicate that temporal aspects of CORT regulation may account in part for differential responses to vibratory stress of CORT-dependent mRNA responses in hippocampus. An increase in GPDH gene activity represents a CORT-dependent stress response that can be used to characterize changes in neuroendocrine status and stress responsiveness of target cells.

Rapid increase in glycerol phosphate dehydrogenase mRNA in adult rat brain: a glucocorticoid-dependent stress response.

Twenty-five years ago, glycerol phosphate dehydrogenase (GPDH, EC 1.1.1.8) was described as a hormonally dependent enzyme in the brain, and since then has been characterized for its developmental regulation and as a marker for oligodendrocytes. These studies describe the cloning of GPDH mRNA from adult rat hippocampus and its characterization as an in vivo response in the brain to both glucocorticoid treatment and stress. A nearly full-length cDNA clone was obtained with sequence homology to the adult mouse GPDH gene. Three EcoRI fragments derived from this clone each hybridized to a major 2.9-kb transcript in poly(A)-containing RNA. GPDH mRNA increased up to 10-fold in a dose-dependent manner in response to acute corticosterone (CORT) treatment (8 h-3 days) of adrenalectomized (ADX) rats. Hybrid-selected GPDH mRNA encodes a 35-kD, pI 6.3 polypeptide that comigrated with our previously described CORT-responsive 35-kD in vitro translation product, with which it shares the same response characteristics. The basal (morning) AM prevalence of GPDH mRNA in the hippocampus is approximately 0.5 pg/micrograms total RNA. Shaking stress increased GPDH mRNA 4-fold; this increase was completely blocked by prior ADX. Hippocampal GPDH mRNA prevalence in ADX rats did not differ from AM intact rats, but increased to stress levels within 2 h of a CORT treatment that produced serum levels in the high physiological or stress range. GPDH expression increased throughout the brain of CORT-treated compared with ADX rats by in situ hybridization; the pattern of expression is similar to that of proteolipid protein mRNA and is consistent with a predominant expression in oligodendrocytes in white matter. Restraint and cold stress also increased GPDH mRNA in the brainstem. These results establish GPDH mRNA as a glucocorticoid-dependent stress response in adult rat hippocampus and indicate that glucocorticoid regulation of GPDH enzyme activity throughout the brain could result from changes in GPDH mRNA prevalence. In addition to its role in development, GPDH may participate in oligodendrocyte responses to stress in the adult brain.

Glucocorticoid receptor mRNA ontogeny in the fetal and postnatal rat forebrain.

Glucocorticoid receptor (GR) ontogeny and distribution in postnatal rat brain have been demonstrated, but onset and distribution of GR gene expression during fetal life has not been reported. This study focuses on the distribution of GR-mRNA in the fetal and postnatal rat forebrain, with emphasis on hypothalamic and limbic structures. Time pregnant rats were decapitated at 8:30-9:30 AM on Gestational Days 14 (F14), F16, F17, F18, and F19. Postnatally, rats were sacrificed on Days 1, 4, 6, 10, and 16. Cryostat sections were subjected to in situ hybridization, using a cRNA probe directed to the GR-mRNA. GR-mRNA was detectible in the hippocamposeptal formation as early as F14. By F16, GR gene expression was evident in the hypothalamic paraventricular nucleus (PVN) as well. During late gestation (F17-F19), GR-mRNA was localized also in the thalamus, hippocampus, amygdala, and discrete cortical regions. Postnatally, GR-mRNA abundance was high in the PVN, CA1/CA2 hippocampal field, piriform cortex and dorsal endopiriform nucleus, specific amygdaloid nuclei, and the suprachiasmatic nucleus. In PVN, GR-mRNA was present prior to the onset of CRH gene expression (F17), which may suggest a role for GR in neuronal differentiation.

Effect of chronic adrenalectomy on neuron loss and distribution of sulfated glycoprotein-2 in the dentate gyrus of prepubertal rats.

This study extends the unexpected finding of Sloviter et al. (Science, 1989, 243: 535-538) that adrenalectomy (ADX) of young rats casues a loss of granule neurons in the dentate gyrus. In particular, we determined how the vulnerability of dentate granule neurons to the cytocidal effect of ADX is related to the completeness of the ADX and whether sulfated glycoprotein-2, a putative component of programmed cell death, is associated with the death of granule neurons after ADX. We report that 4 months after bilateral ADX of young (150-175 g) rats only ADX rats that had attenuated weight gain and less than 2 ng/ml of serum corticosterone lost granule neurons; whereas as little as 15 ng/ml of serum corticosterone was sufficient to protect granule neurons from cell death. In addition, by immunocytochemistry, SGP-2 was distributed as punctate deposits throughout the molecular layer of the dentate gyrus and in glial cells juxtaposed to surviving neurons in the dentate of ADX rats with a granule cell loss. However, immunoreactivity for SGP-2 was not found in granule neurons that exhibited morphological signs of cellular generation after ADX.

Human dihydrofolate reductase gene is located in chromosome 5 and is unlinked to the related pseudogenes.

The chromosomal location of the human dihydrofolate reductase (DHFR; EC 1.5.1.3) gene that is amplified in a methotrexate-resistant human cell line has been investigated by screening a large number of human-mouse cell hybrids containing overlapping subsets of human chromosomes. A correlation of genomic blotting data with the chromosome constitution of the individual cell hybrids has allowed the assignment of the human DHFR gene to chromosome 5. This chromosome assignment has been confirmed by the observation of a concomitant loss of the human DHFR gene and of sensitivity to diphtheria toxin, a marker associated with chromosome 5, in two human-mouse cell hybrids selected for resistance to the toxin. Six EcoRI fragments of human DNA containing DHFR pseudogenes or other DHFR-related sequences have been assigned to chromosomes other than chromosome 5.

Corticosterone-induced responses in rat brain RNA are also evoked in hippocampus by acute vibratory stress.

Corticosterone (CORT) induces responses in brain cells that are mediated by glucocorticoid receptors through regulation of gene activity. We previously found rapid increases in select poly(A)-containing RNAs in rat hippocampus following treatment with CORT that are mediated by low-affinity glucocorticoid receptors. To determine if these responses are hippocampal specific, we examined RNA responses to glucocorticoids in several brain regions, myocardium, and cultured astrocytes by two-dimensional gel electrophoretic resolution of 35S-methionine labelled, in vitro translation products. RNAs coding for similar 35-, 33-, and 20-kdalton polypeptides are induced after 3 days of CORT treatment (40 mg/kg/day) in hippocampus, hypothalamus, cortex, striatum, cerebellum, and myocardium. Primary astrocyte cultures (neonatal rat), however, showed increases after hydrocortisone (1 microgram/ml) in only the 20- and 33-kdalton translation products, while the 35-kdalton polypeptide was not detected. The hippocampal responses were maintained for up to 3 months during chronic daily CORT treatment. To determine if an increase in endogenous CORT levels would also evoke the RNA responses, we subjected rats to 2 h vibratory stress and analyzed the in vitro translation products. RNAs coding for the 35- and 20-kdalton polypeptides were increased 3- to 5-fold in the hippocampus after acute stress in intact rats, but not in stressed adrenalectomized rats. These results suggest a new class of molecular stress responses in brain cells that is glucocorticoid dependent under physiological conditions.