Modulation of a novel RNA in brain neurons by glucocorticoid and mineralocorticoid receptors.

A novel cDNA clone, CR16, was isolated from a rat hippocampal cDNA library and characterized for responses to corticosteroids and regional expression. The 4-kb RNA was increased 3-fold by treatment of adrenalectomized (ADX) rats with corticosterone (CORT). Overlapping cDNA totaling 4,374 nt were used to define an open reading frame of 1,356 nt beginning 191 nt from the 5'-end and encoding a 45-kD protein containing 32% proline. CR16 has no obvious homologies to GenBank or protein databases. CR16 RNA was detected by in situ hybridization in neuron-rich layers of the hippocampal formation, layers II, III and VI of the cerebral cortex, thalamus, ventromedial nucleus of the hypothalamus, bed nucleus of the stria terminalis, lateral septal nucleus, nucleus accumbens, olfactory bulb, inferior colliculus, pons and inferior olive. The CR16 RNA has low prevalence in the hippocampus and cortex (< 10 pg/micrograms total RNA) and is elevated 3-fold in both structures in a dose-dependent manner by CORT in ADX rats. Treatment of ADX rats with aldosterone (ALDO), CORT, or RU28362 increased CR16 RNA to similar levels in the hippocampus while ALDO had minimal effects on the level of CR16 RNA relative to CORT or RU28362 in the cortex. Neither shaking stress (2 h) nor 2 h CORT significantly elevated CR16 RNA in the hippocampus, suggesting that its response to elevated CORT is not rapid. ADX lowered CR16 RNA levels by 50% relative to intact rats while low-level CORT replacement (> or = 4 ng/ml serum CORT) significantly elevated CR16 RNA 2-fold in ADX rats. These results are consistent with both the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) regulating the CR16 gene. This gene will be useful in dissecting the role of MR and GR in CNS neurons.

Clusterin (apoJ) alters the aggregation of amyloid beta-peptide (A beta 1-42) and forms slowly sedimenting A beta complexes that cause oxidative stress.

Clusterin (apoJ), a multifunctional apolipoprotein made by cells in the brain and many other locations, is associated with aggregated amyloid beta-peptide (A beta) in senile and diffuse plaques of Alzheimer's disease (AD). We observed that purified human serum clusterin partially blocked the aggregation of synthetic A beta 1-42, as shown by centrifugal assays (14,000g x 10 min) and by atomic force (scanning probe) microscopy. Slowly sedimenting A beta complexes were formed in the presence of clusterin, which included aggregates > 200 kDa that resist dissociation by low concentrations of SDS. Clusterin enhanced the oxidative stress caused by A beta, as assayed by oxidative stress in PC12 cells with MTT, which is widely used to estimate neurotoxicity. These indications of enhanced neurotoxicity by the MTT assay were observed in the highly aggregated rapidly sedimenting fraction, but also in more slowly sedimenting "soluble" forms. This novel activity of slowly sedimenting A beta may enhance the neurotoxicity of A beta deposits in AD brains, because soluble complexes have a potential for diffusing to damage distal neurons.

Methylation of the rat glial fibrillary acidic protein gene shows tissue-specific domains.

The gene for glial fibrillary acidic protein (GFAP) was compared for CpG sites that are potential locations of methylated cytosine (mC). GFAP sequences in the 5'-upstream promoter and in exon 1 of rat, mouse, and human showed extensive similarity in the locations of CpG sites in the promoter and in exon 1, implying conservation. The methylation of mC at 9 CpG sites in the promoter and 10 sites in exon 1 was analyzed in F344 male rats by a quantitative application of ligation-mediated polymerase chain reaction (LMPCR). CpG sites with varying mC in different tissues were found in the GFAP promoter and in a CpG island in exon 1. In the brain, the promoter had about 40% less mC than in testis and liver. The degree of methylation varied strikingly between adjacent sites within and between tissues. Testis GFAP exon 1 had a gradient of mC from 5' to 3' across the exon that was absent in liver, brain, and cultured neurons and astrocytes. Among brain regions, the hippocampus had 10-40% less mC at 12 CpG sites than in hypothalamus; the other sites (7/19) showed smaller differences between these brain regions. In DNA from primary cultures, astrocytes had slightly less mC than neurons at all sites. Because neuron-rich hippocampal subregions and primary neurons cultures had less methylation than nonneural tissues, we hypothesize that neuroectodermal derivatives tend to be less methylated, whether or not GFAP is expressed. Four domains of methylated CpG sites are proposed on the basis of tissue and cell-type distribution: I) a constitutively methylated domain in the mid-upstream promoter; II) a testis-specific gradient of methylation in exon 1; III) a hypomethylated domain found in neuroectodermal derivatives; and IV) subsets of sites in the promoter and in exon 1 that have the least methylation in astrocytes, and therefore may be astrocyte-specific domains.

Age-related increases in glial fibrillary acidic protein do not show proportionate changes in transcription rates or DNA methylation in the cerebral cortex and hippocampus of male rats.

Age-related increases in the expression of glial fibrillary acidic protein (GFAP) in many brain regions are observed in short- and long-lived mammals. Possible genomic mechanisms for the increase of GFAP mRNA and protein were studied in the hippocampus and cortex of male F344 rats and a longer-lived hybrid F1 (F344 x Brown Norway). No age-related changes were found in the extent of cytosine methylation at 19 CpG sites in the 5'-upstream GFAP promoter and in exon 1. With the nuclear runon assay, no change was found in the transcription rate of GFAP in the cerebral cortex or hippocampus. Thus, age-related increases in GFAP are not associated with proportionate changes in transcription rates or DNA methylation. However, the transcription of glutamine synthetase was increased by about 60%. These findings contrast with age-related loss of bulk tissue DNA methylation and decreased transcription rates of other genes reported in non-neural tissues.

Purification and characterization of brain clusterin.

Clusterin, a 70-80 kDa sulfated glycoprotein found in numerous tissues, is also known as complement lysis inhibitor (CLI), apolipoprotein J, SP-40,40, TRPM-2, and SGP-2. In Alzheimer disease (AD), clusterin mRNA is increased, whereas clusterin protein is found in deposits of beta-amyloid (A beta). These studies characterized clusterin protein from human brain. In extracts from cortex and hippocampus, clusterin was about 40% higher in AD than in controls. Purified clusterin from human brain was slightly smaller than serum clusterin. Brain and serum clusterin were indistinguishable in the inhibition of complement-mediated hemolysis. Both serum and brain clusterin were indistinguishable in inhibiting the aggregation of A beta and promoting oxidative stress in rat pheochromocytoma PC12 cells (MTT assay). The inhibition of A beta aggregation and enhancement of A beta toxicity by clusterin suggest new mechanisms in AD.

Age-dependent increases of DNA adducts (I-compounds) in human and rat brain DNA.

Brain DNA from 20 humans ranging in age from neonatal to 100 years was analyzed by the nuclease P1-enhanced version of the 32P-postlabeling assay for bulky covalently modified nucleotides. A reproducible pattern of three 32P-labeled spots was obtained by thin-layer chromatography followed by autoradiography. Two of these spots increased with age (Mann-Whitney U-test; P < 0.001; comparison of ages < or = 60 years and ages > 60 years). Thus, these spots met the definition of I-compounds. Rat brain DNA exhibited the same two I-spots, whose intensities also increased with animal age (1, 4, and 10 months). In humans, considerable individual variation of brain I-compound levels was observed, especially at ages > 60 years, presumably reflecting environmental, life-style, or genetic factors. This variation was not noted for brain DNA of laboratory rats. Thus, human brain DNA undergoes progressive covalent modifications with aging.

New mRNA probes for hippocampal responses to entorhinal cortex lesions in the adult male rat: a preliminary report.

Three new mRNA responses were found in the hippocampus of the adult male rat after entorhinal cortex lesions (ECL) that induce synaptic reorganization. Hippocampus cDNA libraries were screened by a subtractive hybridization strategy designed to detect ECL-induced mRNAs. Partial sequencing showed clones with similarities to mouse vimentin, ferritin, and polypeptide 7B-2. A sequence similar to mouse SNAP-25 sequence was also detected. Using a mouse SNAP-25 probe, rat SNAP-25 mRNA increased in the hippocampus after ECL.

Slow changes of tyrosine hydroxylase gene expression in dopaminergic brain neurons after neurotoxin lesioning: a model for neuron aging.

Slow neuron regression develops during the adult phase of life in select brain systems of mammals. We describe a model in adult rats that resolves several phases in a slow atrophic process that differentially influences levels of mRNA and protein for tyrosine hydroxylase (TH). Responses of striatal dopaminergic markers to 6-hydroxydopamine (6-OHDA) lesions in rats indicated that the striatal terminals maintained TH protein, despite greater than 3-fold loss of TH mRNA in the substantia nigra pars compacta (SNC) cell bodies whose axons project to the striatum. The loss of TH mRNA/cell was progressive up to 9 months, whereas SNC cell body shrinkage stabilized by 3 months post-lesioning. Consideration of possible mechanisms in protein turnover motivated a search for PEST motifs in the TH of rats and other vertebrates that could be a point of regulation by altering the rate of TH protein turnover.

Differential contributions of ovarian and extraovarian factors to age-related reductions in plasma estradiol and progesterone during the estrous cycle of C57BL/6J mice.

The relative contributions of ovarian and extra-ovarian factors to the altered ovarian steroidal profiles of middle-aged mice were assessed by reciprocal, heterochronic ovarian grafting. Ovaries from cycling, young (2 months), and middle-aged (12 months) mice were exchanged by grafting under the renal capsules. Blood samples were obtained daily at midday throughout the estrous cycle for measurement of estradiol (E2) and 3-4 h after lights-out on proestrus to measure the preovulatory elevation of progesterone (P4). Middle-aged intact mice had lower mean concentrations of E2 during the cycle, no detectable midday preovulatory elevation of E2, and an attenuated preovulatory increase of P4 compared to young mice. Ovarian grafts from young donors failed to increase mean E2 levels of middle-aged mice, but did restore the preovulatory elevation of E2 and preovulatory P4 to levels of young controls. Reciprocal grafting confirmed these findings: ovaries from middle-aged donors in young hosts produced mean E2 levels equivalent to those of young mice but were unable to support a preovulatory increase of E2 or a preovulatory P4 level equivalent to that of young controls. These results reveal differential contributions of ovarian and extra-ovarian factors to age changes in E2 and P4. They indicate that ovarian aging plays an important role in attenuating the preovulatory increase of E2 and P4, but extra-ovarian, presumably neuroendocrine, age changes underlie the mean reduction of E2 levels across the estrous cycle.

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.

Selective alterations of RNA in rat hippocampus after entorhinal cortex lesioning.

In vitro translation products from RNA of rat hippocampus after deafferentation by entorhinal cortex lesions were analyzed by two-dimensional gel electrophoresis. Although hippocampal total RNA yield was not affected 14 days after the lesion, analysis of the gels showed reproducible changes in the steady-state level of several transcripts. Glial fibrillary acidic protein RNA increased 2-fold over control hippocampi RNA. Moreover, seven other transcripts of unknown identity had increased prevalence in the denervated hippocampus. The changes, which ranged from 2- to 20-fold, involved mRNA encoding small slightly acidic polypeptides: 12 kDa (pI 5.6), 13 kDa (pI 6.1), 20 kDa (pI 5.8), 31 kDa (pI 5.7), 33 kDa (pI 5.7), 35 kDa (pI 5.6), and 53 kDa (pI 5.4). These results suggest new molecular markers for analyzing the complex mechanisms of synaptic reorganization in the dentate gyrus after deafferentation.

Oral administration of estradiol to young C57BL/6J mice induces age-like neuroendocrine dysfunctions in the regulation of estrous cycles.

Exogenous estradiol (E2) can accelerate the onset of acyclicity and other age-related neuroendocrine changes in rodents. The present study demonstrates that chronic oral administration of E2 (850 micrograms/kg body wt/day) induces premature acyclicity in intact C57BL/6J mice. After 6 wk of E2, mice regained cyclicity but ceased cycling prematurely, whereas 12 wk of E2 caused permanent acyclicity in all mice. The acyclicity after 12 wk of E2 was not reversed by ovarian replacement from young donors, which implies extra-ovarian (neuroendocrine) lesions. The above studies with intact mice can not identify the contributions from exogenous E2 and endogenous ovarian sections and the possible impact of oral E2 on the ovary. Therefore, mice were ovariectomized (OVX) to remove ovarian contributions, treated for 12 wk with oral E2, and then were given young ovarian grafts and assayed for neuroendocrine functions. Approximately 50% of the E2-treated and grafted mice were permanently acyclic, whereas controls cycled well. Thus, oral E2 causes irreversible neuroendocrine damage. However, the presence of the ovary during E2 treatment increases the loss of cyclicity, implying a dose effect. We conclude that the induction of acyclicity is dependent on the dose and duration of E2 exposure.

Alterations in hypothalamic serotonergic-catecholaminergic relationships in aging C57BL/6J female mice.

The effects of aging and estradiol on the three major hypothalamic monoaminergic systems were measured in female C57BL/6J mice. Both aging and estradiol treatment induced small (10-15%) changes in the levels and ratios of the monoamines and their catabolites. In addition, the levels of 5-hydroxyindoleacetic acid, the major serotonergic catabolite, were strongly correlated with the levels of the two major dopaminergic catabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Aging affected the relationship between 5-hydroxyindoleacetic acid and the dopaminergic catabolites such that the slope of the regression lines of 5-hydroxyindoleacetic acid on the dopaminergic compounds was increased in older mice. The age-related alteration in the correlations between the serotonergic and dopaminergic compounds may be due to an effect of aging on the acid export system.

Hypothalamic monoamines and their catabolites in relation to the estradiol-induced luteinizing hormone surge.

Monoamines and non-conjugated catabolites (serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 3,4-dihydroxyphenyl-acetic acid (DOPAC), homovanillic acid (HVA), 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), norepinephrine (NE), and dopamine (DA] were measured in the medial basal hypothalamus (MBH) and preoptic area (POA) of ovariectomized (OVX) and OVX estradiol (E2)-treated rats using high-performance liquid chromatography with electrochemical detection. These E2 treatments were sufficient to induce an LH surge. The use of MHPG/NE ratios as estimates of NE release was validated in the rat hypothalamus by the major decreases of MHPG after injection of the alpha 2-adrenergic agonist, clonidine, and by MHPG increases after the alpha 2-antagonist, yohimbine. The ratio, MHPG/NE, decreased between morning and afternoon in the MBH but not in the POA; there were no differences between OVX and E2-treated rats. Previous studies using a variety of methods indicate that NE turnover increases during LH surges. The present data suggest that unconjugated MHPG is not a sensitive measure of NE release in the rat hypothalamus, but can detect the large changes produced by stimulating or inhibiting the alpha 2-adrenergic autoreceptor. The ratios of DOPAC/DA and 5-HIAA/5-HT in the MBH decreased consistently between morning and afternoon in OVX rats, with or without E2 treatment. This suggests that the release of DA and 5-HT decreases during the day regardless of steroidal milieu.

Compartmentalization of calmodulin and tubulin in the male C57BL/6J mouse brain: heterogeneity of age changes in calmodulin compartments.

Calmodulin (CaM) and tubulin were analyzed by radioimmunoassay in subcellular fractions prepared from cerebral cortex and striatum of aging male C57BL/6J mice. Three fractions were prepared by a new procedure: cytosol (soluble); EGTA-releasable, membrane-bound; and detergent-extractable (Triton X-100), membrane-bound fractions. CaM concentration in all three fractions prepared from striatum showed small (10-15%, p less than 0.05) decreases with age (3-31 months). Cortical CaM concentrations were less affected by age, and only the EGTA-releasable fraction decreased. To compare functional activity and immunoreactivity of CaM, soluble CaM was also assayed by the activation of cyclic nucleotide phosphodiesterase (PDE). The radioimmunoassay and PDE activation assays gave equivalent results, suggesting that no alteration occurred with age in biological activity of CaM, via post-translational modification or other mechanisms. Soluble and particulate tubulin concentration did not change significantly with age in either brain region. The changes observed in striatal CaM, particularly in membrane-bound compartments, could contribute to the age-related decline in mammalian basal ganglial function.

Alteration of calmodulin distribution does not accompany dopaminergic supersensitization of the mouse striatum.

Membrane-bound calmodulin increases following dopaminergic supersensitization of the rat striatum. To assess the generality of this relationship, mice were treated with two different supersensitization paradigms. Calmodulin levels and subcellular distribution were determined by radioimmunoassay. Chronic haloperidol treatment increased striatal D2 dopamine receptor density by 25% but had no effect on membrane-bound calmodulin levels. Similarly, 6-hydroxy-dopamine (6-OHDA) lesions depleted striatal dopamine content greater than 95% without affecting membrane-bound calmodulin. In contrast, soluble calmodulin levels decreased by 15% in the 6-OHDA-lesioned striatum, suggesting that soluble calmodulin is enriched in presynaptic dopaminergic terminals. We conclude that dopaminergic supersensitization can occur in the mouse striatum in the absence of any change in calmodulin distribution.

Effective oral administration of 17 beta-estradiol to female C57BL/6J mice through the drinking water.

17 beta-Estradiol (E2) was dissolved in the drinking water of female C57BL/6J mice and presented ad libitum. The oral administration of E2 produced expected responses in E2-sensitive target tissues. Vaginal smear cytology changed from the thin leukocytic smears characteristic of ovariectomized controls to thicker smears containing only cornified epithelial cells. Uterine weight and the specific activities of uterine glucose-6-phosphate dehydrogenase and alkaline phosphatase were elevated, while the postovariectomy elevation in serum luteinizing hormone (LH) was suppressed. However, oral E2 did not influence the specific activity of uterine acid phosphatase. During oral administration of E2 through the drinking water, serum estrone and E2 were elevated during the night and returned to low baseline levels during the day, in parallel with the circadian patterns of drinking. Similar transient elevations of serum E2 levels were observed after subcutaneous injections of E2. The oral administration of E2 has advantages over the widely utilized parenteral routes of E2 administration (i.e., injection or surgical implantation of E2-containing capsules), particularly for long-term experiments, and may be more analogous to the usual oral route of estrogen administration in women as contraceptives or as postmenopausal estrogen-replacement therapy.

Elevated density of [3H]imipramine binding in aged human brain.

Aging was associated with an increase in the density of specific binding sites for [3H]imipramine in postmortem specimens of human hypothalamus, frontal cortex, and parietal cortex. In general, [3H]imipramine binding was not affected by factors considered difficult to control in postmortem studies, i.e., time from death to autopsy and cause of death. The in vitro regulation of [3H]imipramine binding by sodium was impaired with age in hypothalamic homogenates. In vitro regulation of [3H]imipramine binding by chloride was intact. Determination of the concentrations of 5-hydroxytryptamine (serotonin) and 5-hydroxyindoleacetic acid in hypothalamus and frontal cortex indicated no apparent age-related changes in indole metabolism. The age-related increase in brain [3H]imipramine binding and impairment in the in vitro regulation of binding by ions are similar to changes observed previously in aged mouse brain. The increase in brain antidepressant binding sites is discussed in relationship to other indices of brain serotonergic function in aging and to the relationship of [3H]imipramine binding and depression.

Catecholamine levels and turnover during aging in brain regions of male C57BL/6J mice.

A radioenzymatic assay is described for measuring brain catecholamines (CA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the same tissue extract. The [3H]-methylated products are differentially extracted and then acetylated by acetic anhydride, followed by thin layer chromatography in non-basic solvents. Routine sensitivity is 3-5 pg per sample. This assay gave CA levels in brain regions which generally agreed with previous reports. CA levels/mg protein and turnover (after injection of a-methyl-p-tyrosine) were measured in male C57BL/6J mice at various ages between 4 and 30 months, the average lifespan of male C57BL/6J mice. No region showed progressive age changes in levels or turnover, or changes before midlife, 8-12 months. Brain regions with no evidence of age changes include globus pallidus, zona incerta, substantia nigra, cerebellum, and olfactory bulbs. Small changes (10-25%) were detected in only some regions of mice aged greater than or equal to 24 months; few changes were statistically significant. CA levels and turnover decreased in some samplings of striatum and median eminence-arcuate nucleus. In contrast, DA levels increased in anterior pituitary and possibly in medial preoptic region. DOPAC levels (measured only in rostral striatum) decreased by 20% at 28 months. These results diverge from the larger, progressive decreases of DA reported in human striatum during aging.