Short-Term Memory Deficit Associates with miR-153-3p Upregulation in the Hippocampus of Middle-Aged Mice.

The early stages of ageing are a critical time window in which the ability to detect and identify precocious molecular and cognitive markers can make the difference in determining a healthy vs unhealthy course of ageing. Using the 6-different object task (6-DOT), a highly demanding hippocampal-dependent recognition memory task, we classified a population of middle-aged (12-month-old) CD1 male mice in Impaired and Unimpaired based on their short-term memory. This approach led us to identify a different microRNAs expression profile in the hippocampus of Impaired mice compared to Unimpaired ones. Among the dysregulated microRNAs, miR-153-3p was upregulated in the hippocampus of Impaired mice and appeared of high interest for its putative target genes and their possible implication in memory-related synaptic plasticity. We showed that intra-hippocampal injection of the miR-153-3p mimic in adult (3-month-old) mice is sufficient to induce a short-term memory deficit similar to that observed in middle-aged Impaired mice. Overall, these findings unravel a novel role for hippocampal miR-153-3p in modulating short-term memory that could be exploited to prevent early cognitive deficits in ageing.

MicroRNA-335-5p modulates spatial memory and hippocampal synaptic plasticity.

MicroRNAs are endogenous, noncoding RNAs crucial for the post-transcriptional regulation of gene expression. In this study, we investigated the role of miR-335-5p in spatial learning and synaptic plasticity. To this end we first showed spatial learning induced down-regulation of miR-335-5p. Next we found impairment in long-term memory and reduction in hippocampal long-term potentiation by exogenous administration of the miRNA. These findings demonstrate that miR-335-5p is a key coordinator of the intracellular pathways that mediate experience-dependent changes in the brain.

microRNAs Modulate Spatial Memory in the Hippocampus and in the Ventral Striatum in a Region-Specific Manner.

MicroRNAs are endogenous, noncoding RNAs crucial for the post-transcriptional regulation of gene expression. Their role in spatial memory formation, however, is poorly explored. In this study, we analyzed learning-induced microRNA expression in the hippocampus and in the ventral striatum. Among miRNAs specifically downregulated by spatial training, we focused on the hippocampus-specific miR-324-5p and the ventral striatum-specific miR-24. In vivo overexpression of the two miRNAs demonstrated that miR-324-5p is able to impair memory if administered in the hippocampus but not in the ventral striatum, while the opposite is true for miR-24. Overall, these findings demonstrate a causal relationship between miRNA expression changes and spatial memory formation. Furthermore, they provide support for a regional dissociation in the post-transcriptional processes underlying spatial memory in the two brain structures analyzed.

Molecular recognition of amino acids by RNA aptamers: the evolution into an L-tyrosine binder of a dopamine-binding RNA motif.

We report the evolution of an RNA aptamer to change its binding specificity. RNA aptamers that bind the free amino acid tyrosine were in vitro selected from a degenerate pool derived from a previously selected dopamine aptamer. Three independent sequences bind tyrosine in solution, the winner of the selection binding with a dissociation constant of 35 microM. Competitive affinity chromatography with tyrosine-related ligands indicated that the selected aptamers are highly L-stereo selective and also recognize L-tryptophan and L-dopa with similar affinity. The binding site was localized by sequence comparison, analysis of minimal boundaries, and structural probing upon ligand binding. Tyrosine-binding sites are characterized by the presence of both tyrosine (UAU and UAC) and termination (UAG and UAA) triplets.

In vitro selection of dopamine RNA ligands.

RNA aptamers that specifically bind dopamine have been isolated by in vitro selection from a pool of 3.4 x 10(14) different RNA molecules. One aptamer (dopa2), which dominated the selected pool, has been characterized and binds to the dopamine affinity column with a dissociation constant of 2.8 microM. The specificity of binding has been determined by studying binding properties of a number of dopamine-related molecules, showing that the interaction with the RNA might be mediated by the hydroxyl group at position 3 and the proximal aliphatic chain in the dopamine molecule. The binding domain was initially localized by boundary experiments. Further definition of the dopamine binding site was obtained by secondary selection on a pool of sequences derived from a partial randomization of the dopa2 molecule. Sequence comparison of a large panel of selected variants revealed a structural consensus motif among the active aptamers. The dopamine binding pocket is built up by a tertiary interaction between two stem and loop motifs, creating a stable framework in which five invariant nucleotides are precisely arrayed. Minimal active sequence and key nucleotides have been confirmed by the design of small functional aptamers and mutational analysis. Enzymatic probing suggests that the RNA might undergo a conformational change upon ligand binding that stabilizes the proposed tertiary structure.

The relative expression of human histone H2A genes is similar in different types of proliferating cells.

To help elucidate the factors regulating the expression of histone multigene families in proliferating cells, we asked whether the relative expression of different members of such a family was dependent upon or independent of the type of proliferating cell. This question was examined by measuring the relative expression of seven members of the human histone H2A multigene family in four cell lines of diverse origin. Two previously uncharacterized members of the H2A gene family were found to be the most abundantly expressed of the seven in all four cell lines. One of these encodes an H2A.2 species containing methionine. The lines examined in the study were Jurkat (a lymphoma line), N-tera (a pluripotent embryonic carcinoma line), HeLa (originally isolated as a cervical carcinoma), and IMR90 (a normal embryonic fibroblastic line). The amount of each mRNA species was quantitated using oligonucleotides about 30 bases long complementary to the 5' or 3' untranslated regions. In each cell line, there was at least an eight-fold difference in the amount of the most and least highly expressed of the seven H2A mRNA species. In addition, there were up to five-fold differences among the cell lines in the amount of the H2A mRNA species as a fraction of total RNA. However, in contrast to those differences, the four cell lines were found to express the seven H2A mRNAs in similar relative amounts. These findings suggest that the relative expression of the individual members of a histone gene family is independent of the type of replicating cell.

Histone H2A.X gene transcription is regulated differently than transcription of other replication-linked histone genes.

Histone H2A.X is a replication-independent histone H2A isoprotein species that is encoded by a transcript alternatively processed at the 3' end to yield two mRNAs: a 0.6-kb mRNA ending with the stem-loop structure characteristic of the mRNAs for replication-linked histone species, and a second, polyadenylated 1.6-kb mRNA ending about 1 kb further downstream (C. Mannironi, W. M. Bonner, and C. L. Hatch, Nucleic Acids Res. 17:9113-9126, 1989). Of the two, the 0.6-kb H2A.X stem-loop mRNA predominates in many cell lines, indicating that the presence of two types of mRNA may not completely account for the replication independence of H2A.X protein synthesis. The ambiguity is resolved by the finding that the level of the 0.6-kb H2A.X mRNA is only weakly downregulated during the inhibition of DNA replication and only weakly upregulated during the inhibition of protein synthesis, while the levels of other replication-linked mRNAs are strongly down- or upregulated under these two conditions. Analysis of the nuclear transcription rates of specific H2A genes showed that while the rates of transcription of replication-linked H2A genes decreased substantially during the inhibition of DNA synthesis and increased substantially during the inhibition of protein synthesis, the rate of H2A.X gene transcription decreased slightly under both conditions. These differences in transcriptional regulation between the H2A.X gene and other replication-linked histone genes are sufficient to account for the differences in regulation of their respective stem-loop mRNAs.

H2A.X. a histone isoprotein with a conserved C-terminal sequence, is encoded by a novel mRNA with both DNA replication type and polyA 3' processing signals.

A full length cDNA clone that directs the in vitro synthesis of human histone H2A isoprotein H2A.X has been isolated and sequenced. H2A.X contains 142 amino acid residues, 13 more than human H2A.1. The sequence of the first 120 residues of H2A.X is almost identical to that of human H2A.1. The sequence of the carboxy-terminal 22 residues of H2A.X is unrelated to any known sequence in vertebrate histone H2A; however, it contains a sequence homologous with those of several lower eukaryotes. This homology centers on the carboxy-terminal tetrapeptide which in H2A.X is SerGlnGluTyr. Homologous sequences are found in H2As of three types of yeasts, in Tetrahymena and Drosophila. Seven of the nine carboxy-terminal amino acids of H2A.X are identical with those of S. cerevisiae H2A.1. It is suggested that this H2A carboxy-terminal motif may be present in all eukaryotes. The H2A.X cDNA is 1585 bases long followed by a polyA tail. There are 73 nucleotides in the 5' UTR, 432 in the coding region, and 1080 in the 3' UTR. Even though H2A.X is considered a basal histone, being synthesized in G1 as well as in S-phase, and its mRNA contains polyA addition motifs and a polyA tail, its mRNA also contains the conserved stem-loop and U7 binding sequences involved in the processing and stability of replication type histone mRNAs. Two forms of H2A.X mRNA, consistent with the two sets of processing signals were found in proliferating cell cultures. One, about 1600 bases long, contains polyA; the other, about 575 bases long, lacks polyA. The short form behaves as a replication type histone mRNA, decreasing in amount when cell cultures are incubated with inhibitors of DNA synthesis, while the longer behaves as a basal type histone mRNA.

Changes in the synthesis of histone H1(0) and H1 in rat FRTL-5 thyroid cells exposed to thyrotropin.

In absence of thyrotropin (TSH), FRTL-5 rat thyroid cells stop proliferating and lose the functional characteristics of thyroid tissue. FRTL-5 cells regain their differentiated state and their proliferation activity upon addition of TSH. In this study we investigated the synthesis of histone H1 variants and H19(0) in FRTL-5 cells exposed to 10(-8) M TSH, two days after TSH withdrawal. TSH induced the synthesis of some H1 variants and H1. This effect was already evident six hours after TSH addition, thus well before proliferation, DNA or thyroglobulin synthesis was induced. These data indicate that the induction of H1(0) and some H1 variants is an early event after TSH stimulation and may thus be related to the functional differentiation of FRTL-5 cells.

Doxorubicin induces the acetylation of histone H1 in a human colon cancer cell line (LoVo/DX) selected for resistance to the drug, but not in the sensitive parental line (LoVo).

The effect of doxorubicin (DX) treatment on H1 synthesis and acetylation was studied in two human colon adenocarcinoma cell lines, sensitive (LoVo) and resistant (LoVo/DX) to this drug. Histone variants were resolved by a high resolution two-dimensional gel electrophoresis system coupled to fluorography for the detection of radioactive incorporation. The relative synthesis of H1.4 and H1.5 variants was slightly reduced by DX. This is probably related to the inhibition of DNA synthesis consequent to drug treatment. The main effect is that DX induces the acetylation of H1 isoproteins in the LoVo/DX resistant line but not in the parental line, which is 30 times more sensitive to anthracyclines. The different behavior of the two cell lines cannot be attributed to different cellular drug retention since the DX doses chosen (1.25 for LoVo and 40 micrograms/ml for LoVo/DX cells) correspond to similar intracellular drug concentrations. H1 acetylation persisted after exposure to cycloheximide in DX treated LoVo/DX cells, indicating that it is a postranslational event. The induction of H1 acetylation appears rather specific since no increase was found in 3H-acetate incorporation on the total cellular TCA-precipitable fraction. In addition DX treatment did not modify the acetylation of core histones in either LoVo or LoVo/DX cell lines.

Comparison of histone variant synthesis in human lymphocytic leukemia cells and in normal lymphocytes.

The synthesis of core histone variants and of histone H1 variants was determined in fresh leukemic cells of eight patients with leukemia [seven acute lymphoblastic (ALL) and one chronic lymphocytic (CLL)], in normal lymphocytes from healthy donors or from ALL patients in complete remission. Histone variant synthesis was evaluated by incubating cells with [14C]Lys and [3H]Arg in medium without Lys and Arg and then by two-dimensional polyacrylamide gel electrophoretic separations (acetic acid-urea-Triton x-100 acetic acid-urea-hexadecyltrimethylammonium bromide for core histone variants; sodium dodecyl sulfate/acetic acid-urea-hexadecyltrimethyl ammonium bromide for H1 variants). As previously reported, quiescent lymphocytes and lymphocytes stimulated with phytohaemagglutinin (PHA) showed clearcut changes in the proportions of synthesis of core histone variants and H1 variants. Leukemic lymphocytes freshly obtained from blood showed a pattern of core histone synthesis and H1 synthesis intermediate between that of quiescent and PHA-stimulated lymphocytes; this is probably due to the presence of a mixture of resting and growing cells. When leukemic cells were stimulated to grow by mitogens, the pattern of core histone and H1 variant synthesis was similar to that in mitogen-stimulated normal lymphocytes. Histone variants whose synthesis is associated with the S-phase were not synthesized in leukemic cells treated with the DNA synthesis inhibitors hydroxyurea and 1-beta-D-arabinofuranosylcytosine (Ara-C). The pattern of acetylation of histone H4 was also apparently similar in leukemic cells and normal lymphocytes. The radioactivity associated with the ubiquitinated forms of H2A increased in nongrowing lymphocytes and in leukemic cells treated with DNA synthesis inhibitors whereas they decreased after mitogenic stimulation. Variability was wide in the synthesis of ubiquitinated H2A in different cases of leukemia. The only clear-cut difference between leukemic cells and normal lymphocytes was that leukemic cells from ALL patients, but not lymphocytes from normal donors or from ALL patients in complete remission, synthesized appreciable amounts of H1 degrees, increasing after hydroxyurea/Ara-C treatment and decreasing after PHA-stimulation. In leukemic cells from a CLL patient H1 degrees synthesis was undetectable.

Characterization of transformed cell lines evolving from a normal C3H line.

The development of transformed cell lines evolving from an embryo fibroblastic C3H primary culture was followed before and after the ageing crisis using different techniques. By flow cytometry, alteration of subpopulations having different DNA content and altered metabolic activity was observed after the crisis, with the trend to assume a near tetraploid DNA index at higher passages. The fibrin clot retractile activity was lost in all cases during the ageing crisis, but the outcome did not present uniform values of growth characteristics or chromosome number and tumorigenicity appeared to be a nonstable property of the transformed cell lines.

Histone H1(0) is synthesized by human lymphocytic leukemia cells but not by normal lymphocytes.

Using a two-dimensional gel electrophoresis system (sodium dodecyl sulphate/acetic acid-urea-hexadecyltrimethylammonium bromide) coupled with fluorography, we investigated the synthesis of H1 isoproteins in leukemic cells obtained from peripheral blood of eight children suffering from acute lymphoblastic leukemia (ALL) (four T-ALL, three common ALL, and one B-ALL) and in normal peripheral lymphocytes. H1 was synthesized in leukemic cells but not in normal lymphocytes. Inhibition of DNA synthesis with hydroxyurea and 1-beta-D-arabinofuranosylcytosine induced an increase of relative synthesis of H1(0) in leukemic cells but did not induce any detectable synthesis of H1 in normal lymphocytes. H1(0) synthesis was also undetectable in peripheral lymphocytes of leukemic children in complete remission after chemotherapy and in lymphocytes of a homozygotic twin of a leukemic patient. H1(0) may be a marker of malignant transformation of lymphocytes and as such could be of use for early detection of relapse in patients with acute lymphocytic leukemia in apparent complete remission.

Study of the optimal temperature for the liver microsomal assay with mice S9 fractions.

The effect of temperature on enzymatic activity and stability was studied with respect to the monooxygenase activities of aminopyrine-N-demethylase (APD) and p-nitroanisole O-demethylase (pNAD) under incubation conditions for the liver microsomal assay. The activities of S9 liver fractions of mice induced with sodium phenobarbital and beta-naphthoflavone were determined during a period of preincubation in a range of temperatures from 30 to 44 degrees C. The greatest value of the mean specific activity was found at 40-42 degrees C for both APD and pNAD. The rapid increase of lipid peroxidation after 1 h of incubation at temperatures higher than 42 degrees C can provide an explanation of the enhancement of the rate of inactivation. In order to determine whether biological response is affected by the modifications induced by temperature in the metabolic activating system, tester strain D7 of Saccharomyces cerevisiae was used to assay the genetic activity of the well known premutagenic agent cyclophosphamide by incubating the mixtures both at the traditional temperature of 37 degrees C and at 42 degrees C. We suggest that the use of more favourable conditions for LMA with respect to enzymatic activity, than the traditional ones could improve the reliability and the sensitivity of such tests.