mRNA Levels In Animals Research Articles

MicroRNA Mechanisms of Action: What have We Learned from Mice?

MicroRNAs (miRNAs) are endogenously encoded single-stranded RNAs of about 22 nucleotides (nts) in length that play essential roles in a large variety of physiological processes in animals and plants (Ambros, 2004; Bushati and Cohen, 2007). Mature miRNAs are integrated into the RNA-induced silencing complex (RISC), whose core component is one of the Argonaute family proteins. MiRNAs then direct RISCs to target mRNAs, which are recognized through partial sequence complementarity. Bioinformatic prediction and experimental target gene identification have shown that a miRNA binds mRNAs of hundreds of protein coding genes, which often span a broad spectrum of functional categories (Bartel, 2009; Chi et al., 2009; Hafner et al., 2010). The functional consequence of miRNA-target mRNA interaction and the mechanism of miRNA action have been under intensive investigation and remain a matter of hot debate. It was initially thought that miRNAs repress the protein output of a small number of target genes without significantly affecting their mRNA levels in animals (Lee et al., 1993; Wightman et al., 1993). Subsequent genetic studies in C. elegans and zebrafish showed that miRNAs promote the degradation of their target mRNAs (Bagga et al., 2005; Giraldez et al., 2006). Later, a series of genome-wide studies of in vitro cultured mammalian cell lines transiently transfected with chemically synthesized miRNA mimics led to the conclusion that the predominant functional consequence of miRNA action is target mRNA degradation (Guo et al., 2010). A follow-up study employing temporal dissection of zebrafish development seems to reconcile these two opposite observations by revealing that translational repression precedes target mRNA decay, and suggesting that the immediate outcome of miRNA-target mRNA interaction is translation inhibition but mRNA degradation can follow (Bazzini et al., 2012). Similarly, re-analysis of the previous datasets from cultured cell lines transiently transfected with synthetic miRNA mimics also found that translation repression precedes mRNA degradation (Larsson and Nadon, 2013).

Snca and Bdnf Gene Expression in the VTA and Raphe Nuclei of Midbrain in Chronically Victorious and Defeated Male Mice

BackgroundAlpha-synuclein (α-Syn) is a small neuronal protein that has been found to be expressed throughout the brain. It has been shown that α-Syn regulates the homeostasis of monoamine neurotransmitters and is involved in various degenerative and affective disorders. There is indication that α-Syn may regulate expression of the brain-derived neurotropic factor (BDNF) which plays an important role in the mood disorders.Methodology/Principal FindingsThe study aimed to analyze the mRNA levels of Snca and Bdnf genes in the ventral tegmental area (VTA) and raphe nuclei of the midbrain in male mice that had each won or defeated 20 encounters (20-time winners and 20-time losers, respectively) in daily agonistic interactions. Groups of animals that had the same winning and losing track record followed by a no-fight period for 14 days (no-fighting winners and no-fighting losers) were also studied. Snca mRNA levels were increased in the raphe nuclei in the 20-time losers and in the VTA of the 20-time winners. After no-fight period Snca mRNA levels decreased in both groups. Snca mRNA levels were similar to the control level in the VTA of the 20-time losers and in the raphe nuclei of the 20-time winners. However Snca gene expression increased in these areas in the no-fighting winners and no-fighting losers in comparison with respective mRNA levels in animals before no-fight period. Bdnf mRNA levels increased in VTA of 20-time winners. Significant positive correlations were found between the mRNA levels of Snca and Bdnf genes in the raphe nuclei.Conclusions/SignificanceSocial experience affects Snca gene expression depending on brain areas and functional activity of monoaminergic systems in chronically victorious or defeated mice. These findings may be useful for understanding the mechanisms of forming different alpha-synucleinopathies.

Green tea increases anti-inflammatory tristetraprolin and decreases pro-inflammatory tumor necrosis factor mRNA levels in rats

BackgroundTristetraprolin (TTP/ZFP36) family proteins have anti-inflammatory activity by binding to and destabilizing pro-inflammatory mRNAs such as Tnf mRNA, and represent a potential therapeutic target for inflammation-related diseases. Tea has anti-inflammatory properties but the molecular mechanisms have not been completely elucidated. We hypothesized that TTP and/or its homologues might contribute to the beneficial effects of tea as an anti-inflammatory product.MethodsQuantitative real-time PCR was used to investigate the effects of green tea (0, 1, and 2 g solid extract/kg diet) on the expression of Ttp family genes (Ttp/Tis11/Zfp36, Zfp36l1/Tis11b, Zfp36l2/Tis11d, Zfp36l3), pro-inflammatory genes (Tnf, Csf2/Gm-csf, Ptgs2/Cox2), and Elavl1/Hua/Hur and Vegf genes in liver and muscle of rats fed a high-fructose diet known to induce insulin resistance, oxidative stress, inflammation, and TNF-alpha levels.ResultsTtp and Zfp36l1 mRNAs were the major forms in both liver and skeletal muscle. Ttp, Zfp36l1, and Zfp36l2 mRNA levels were more abundant in the liver than those in the muscle. Csf2/Gm-csf and Zfp36l3 mRNAs were undetectable in both tissues. Tea (1 g solid extract/kg diet) increased Ttp mRNA levels by 50–140% but Tnf mRNA levels decreased by 30% in both tissues, and Ptgs2/Cox2 mRNA levels decreased by 40% in the muscle. Tea (2 g solid extract/kg diet) increased Elavl1/Hua/Hur mRNA levels by 40% in the liver but did not affect any of the other mRNA levels in liver or muscle.ConclusionThese results show that tea can modulate Ttp mRNA levels in animals and suggest that a post-transcriptional mechanism through TTP could partially account for tea's anti-inflammatory properties. The results also suggest that drinking adequate amounts of green tea may play a role in the prevention of inflammation-related diseases.

Compulsive exercise acutely upregulates rat hippocampal brain-derived neurotrophic factor

This study was to examine the effects of treadmill exercise on the expression of brain-derived neurotrophic factor (BDNF) in rat hippocampus. After 1-wk treadmill familiarization, animals in exercise groups received a 4-wk exercise training or an acute exercise. They were sacrificed 2 h or 2 d after exercise and their hippocampal BDNF mRNA and protein levels were determined. We demonstrated that 1) hippocampal BDNF mRNA and protein levels were both elevated in response to exercise training at 2 h after the last run but not after 2 d; 2) an acute moderate exercise (1 or 3 d) increased BDNF protein levels; 3) acute severe exercise increased BDNF protein and mRNA levels in animals under a familiarization regimen, while suppressed the BDNF mRNA level in rats without treadmill familiarization, paralleling the stress effect of immobilization/water exposure. We conclude that compulsive treadmill exercise with pre-familiarization acutely upregulates rat hippocampal BDNF gene expression.

Postnatal handling does not normalize hypothalamic corticotropin-releasing factor mRNA levels in animals prenatally exposed to ethanol

Postnatal handling has been shown to attenuate some of the deficits in developmental outcome observed following prenatal ethanol exposure (E) although it appears to be ineffective at ameliorating the hypothalamic–pituitary–adrenal (HPA) hyperresponsiveness to stressors that has been observed in adult E animals. However, the effects of postnatal handling on central regulation of HPA activity in E animals, particularly with regard to alterations in steady-state hypothalamic corticotropin-releasing factor (CRF) activity, have not been examined. In the present study, offspring from E, pair-fed (PF), and ad-libitum-fed control (C) groups were exposed to daily handling during the first 2 weeks of life (H) or were left entirely undisturbed until weaning (NH). Basal CRF and arginine vasopressin (AVP) mRNA in the parvocellular portion of the paraventricular nucleus (pPVN) of the hypothalamus were assessed at 90–110 days of age. Prenatal ethanol exposure resulted in elevated basal pPVN CRF mRNA levels compared to those in ad-libitum-fed controls. Handling altered CRF mRNA levels in a sex-specific and prenatal treatment-specific manner. Females showed no significant effects of handling. In contrast, handling decreased CRF mRNA levels in PF and C but not E males compared to their NH counterparts. There were no effects of prenatal ethanol or postnatal handling on AVP mRNA levels. These findings indicate that prenatal ethanol exposure results in elevated basal CRF mRNA levels in adulthood and that handling appears to be ineffective in normalizing those elevations, supporting the suggestion that altered basal HPA regulation in E animals may, at least in part, underlie their HPA hyperresponsiveness to stressors.

Differential Regulation of Cathepsin B and Prorenin Gene Expression in Renal Juxtaglomerular Cells

Cathepsin B (cathB) is a lysosome cysteine proteinase. It has been suggested to process prorenin to renin in the renin producing juxtaglomerular (JG) cells of the afferent arterioles (AA) of the kidney. Whether cathB expression is regulated similarly to prorenin production is yet unknown. We have measured prorenin and cathB mRNA levels as well as cathB protein levels in renal AA of Sprague-Dawley rats subjected to low (0.02% w/w) salt diet and ramipril treatment (10 mg/kg/day), or to normal (0.6%) or high (4%) salt diet. Prorenin and cathB expression were also analyzed in the JG cell line As4.1. Prorenin mRNA levels in animals on normal (plus ramipril), low or high salt diet correlated as 1:10:0.5, respectively, while cathB mRNA levels correlated as 1:1:0.6, respectively. Treatment of the As4.1 cells with 100 nM phorbol-12-myristate-13-acetate (PMA) for 16 h inhibited prorenin mRNA expression 3-fold relative to the control conditions. CathB mRNA abundance was not different between the PMA treated and the control As4.1 cells. Western analysis of the cathB protein abundance has shown no difference between the rats on normal and low salt diet, and decrease by 50% in the rats on high salt diet. The results of this study suggest that prorenin and cathB gene expression in renal JG cells are differentially regulated.

The effect of estrogen on cadmium distribution in rainbow trout ( Oncorhynchus mykiss)

The effects of injections of 17β-estradiol (E2) and Cd, on the distribution of Cd and the induction of metallothionein (MT) mRNA and vitellogenin mRNA was investigated. Bone and liver were the main organs accumulating Cd. However, E2 redirected the metal accumulation from the bone and liver to the gill, gut and muscle upon exposure to E2. Cd did not induce the hepatic MT mRNA levels in animals treated with E2. The VTG mRNA levels were also reduced following co-injection of E2 and Cd. However, the kidney responded to Cd exposure by upregulating MT mRNA even in the presence of E2 treatment. In the liver the reduced MT mRNA induction led to a redistribution of CD from MT to non-MT proteins. The toxicological significance of these alterations in Cd handling remains to be determined.

Time‐Dependent Changes in the Expression of Synapsin I mRNA in the Rat Hippocampus During Long‐Term Potentiation: Comparison with the Changes After Amygdaloid Kindled Seizures

Purpose: Nonepileptogenic high‐frequency stimulation of the perforant path input to the dentate gyrus can result in a long‐lasting increase of synaptic efficacy known as long‐term potentiation (LTP). LTP is the most widely used paradigm for studying the cellular and molecular events underlying neuronal plasticity. The synapsins, a family of phosphoproteins associated with the cytoplasmic surface of the synaptic vesicular membrane, are involved in synaptic maturatioin and regulation of neurotransmitter release. During depolarization, activation of calmodulin kinase II leads to phosphorylation of synapsin I, which weakens the linkage between the cytoskeleton and synaptic vesicles, thereby promoting the availability of synaptic vesicles for exocytosis. To investigate the roles of synapsin 1 in neuronal plasticity, we studied the time‐dependent changes in synapsin 1 messenger RNA (mRNA) expression after hippocampal LTP and compared them with those after kindled seizures. Methods: LTP was induced in the right dentate gyrus of the hippocampus by stimulating the ipsilateral perforant path in 49 rats under urethane anesthesia. LTP was evaluated in terms of the precentage increases in the slope of the excitatory postsynaptic potential (EPSP) and height of the population spike (PS) before brain removal and before the application of LTP‐inducing trains. Rat brains were removed at various times [immediately (time 0), 15 and 30 min, and 1, 2, 4, and 8 h; n = 7 at each time] after the completion of LTP stimulation. For in situ hybridization (ISH), the frozen brains were cut into 10‐μm‐thick sections, and tissue sections were processed by using a 35S‐labeled oligonucleotide antisense probe for synapsin 1 mRNA. The ISH procedures were performed as described previously (Epilepsia 1996;37:6–14). One‐way analysis of variance was used for statistical analysis. Results: LTP‐inducing stimulations produced long‐lasting significant increases in the EPSP slope and PS height for ≤8 h after stimulation, compared with the values just before stimulation. After LTP induction, synapsin 1 mRNA expression in the granule cell layer of the dentate gyrus ipsilateral to the site of stimulation increased significantly in a time‐dependent manner. From 2 to 8 h after stimulation, the levels of synapsin 1 mRNA in the ipisilateral dentate gyrus were significantly higher than those in controls subjected to a sham procedure (n = 6). The synapsin 1 mRNA levels reached a maximum (157.4 ± 7.1% of the control level, mean ± SEM) 8 h after stimulation. The synapsin 1 mRNA levels in animals that received only test pulses (n = 5) did not increase significantly in comparison with the control levels. The time‐dependent increases in synapsin 1 mRNA during LTP were quite consistent with those after amygdaloid kindled seizures [Epilepsia 1997;38(suppl 6):59]. Conclusions: The increase in synaptic proteins was considered to indicate transsynaptic propagation of plasticity and modification of synaptic function in the neural networks, supporting the idea that the increase in synapsin 1 is a marker of enhanced synaptic activity. The findings suggest that the increased level of synapsin 1 mRNA is related to the persistent enhancement of synaptic activity within the neural networks, where the dentate granule cells participate in LTP and kindling.

Glucocorticoids induce corticosteroid-binding globulin biosynthesis by immature mouse liver and kidney.

Marked changes in mouse corticosteroid-binding globulin (CBG) gene expression in the liver and kidney occur postnatally. To study the influence of glucocorticoids on the initiation of mouse CBG biosynthesis in these tissues during the first two weeks after birth, we administered dexamethasone (0.5 microgram/g body wt/day) to 4- and 11-day-old pups for three days. This resulted in higher serum CBG and hepatic CBG mRNA levels in animals, irrespective of their ages. Higher relative amounts of CBG mRNA in the kidneys of 14-day-old pups after three days of dexamethasone treatment co-incided with higher amounts of intact and proteolytically cleaved CGB in their urine, and both are indicative of increased CGB production by the developing renal tubules. When an additional group of 11-day-old pups (n = 4) was treated with 0.25 microgram dexamethasone/g body weight per day for five days, this also resulted in significantly higher levels of serum CBG (P < 0.01), hepatic CBG mRNA (P < 0.01) and renal CBG mRNA (P < 0.05), compared to littermates treated with the oil vehicle alone. In contrast, serum CBG levels progressively decreased in adult female mice during five days of treatment with 0.5 microgram dexamethasone/g body weight per day. Taken together, these data indicate that glucocorticoids induce murine CBG gene expression in the immature liver and kidney, and support the concept that the effects of glucocorticoids on CBG gene expression are developmentally stage-specific.

The peroxisome proliferator-activated receptor regulates mitochondrial fatty acid oxidative enzyme gene expression.

Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes a pivotal reaction in mitochondrial fatty acid (FA) beta-oxidation. To examine the potential role of FAs and their metabolites in the regulation of MCAD gene expression, we measured MCAD mRNA levels in animals fed inhibitors of mitochondrial long-chain FA import. Administration of carnitine palmitoyltransferase I inhibitors to mice or rats resulted in tissue-limited increases in steady-state MCAD mRNA levels. HepG2 cell cotransfection experiments with MCAD promoter reporter plasmids demonstrated that this was a transcriptional effect mediated by the peroxisome proliferator-activated receptor (PPAR). The activity mapped to a nuclear receptor response element that functioned in a heterologous promoter context and specifically bound immunoreactive PPAR in rat hepatic nuclear extracts, confirming an in vivo interaction. PPAR-mediated transactions of this promoter and element were also induced by exogenously added FA and fibric acid derivatives. Induction of PPAR transactivation by perturbation of this discrete metabolic step is unusual and indicates that intracellular FA metabolites that accumulate during such inhibition can regulate MCAD expression and are likely candidates for PPAR ligand(s). These results dictate an expanded role for the PPAR in the regulation of FA metabolism.

Alterations in cytochrome P-450 levels in adult rats following neonatal exposure to xenobiotics.

Neonatal exposure to certain xenobiotics has been shown to alter hepatic metabolism in adult rats in a manner that indicates long-term changes in enzyme regulation. Previously, we have observed changes in adult testosterone metabolism and in cytochrome P-450 (P-450) mRNA levels in animals neonatally exposed to phenobarbital (PB) or diethylstilbestrol (DES). In order to test for other enzyme alterations, we used Western blot procedures for specific P-450s to analyze hepatic microsomes from adult rats (24 wk old) that had been exposed neonatally to DES, PB, 7,12-dimethylbenz[a]anthracene (DMBA), or pregnenolone 16 alpha-carbonitrile (PCN). The most striking effects were observed in the DES-treated males: P-4502C6 and an immunologically similar protein were increased 60 and 90%, respectively, relative to control values, but P-4503A2 was decreased by 44%. No changes were observed in the DES-treated males in levels of P-4502E1, P-4502B, or the male-specific P-4502C13. Adult males neonatally treated with PB had 150% increase in levels of anti-P4502B-reactive protein without significant changes in the other enzymes. The DES- and DMBA-treated females had increased levels of the female-specific P-4502C12 of 38 and 48%, respectively, but no other observed alterations. The results confirm that neonatal exposure to DES or PB can cause alterations in adult hepatic cytochrome P-450 levels but show that these chemicals act on different enzymes. Neonatal DMBA resulted in changes in adult females similar to those produced by the synthetic estrogen DES, but did so at about two-thirds lower dose.