Murine microRNAs implicated in liver functions and aging process

Small non-coding microRNAs (miRNAs) are involved in gene regulation in various cellular and developmental processes, including mechanisms of aging. Here, the mouse liver was used as a paradigm for the study of miRNAs implicated in the aging process in mammals. Expression profiling of 367 murine miRNAs (Sanger Version 8.2) was assessed in livers from 4 to 33 months old mice, and their predicted targets were compared with proteomic profiling data generated from the same animals. Gradual increases in the levels of miR-669c and miR-709 were observed from mid-age of 18-33 months, while miR-93 and miR-214 were significantly up-regulated only in extremely old liver. In contrast, we did not identify any miRNAs showing significant down-regulation with age. Interestingly, the up-regulated miRNAs' targets are associated with detoxification activity and regeneration capacity functions known to decline in old liver. In particular, three up-regulated miRNAs may contribute to the aging-related decline in oxidative defense by targeting various classes of glutathione S-transferases. Other proteins in decline in old liver and targeted by the up-regulated miRNAs are involved in mitochondrial functions or maintenance. Taken together, we identified the up-regulation of key miRNAs that may participate in the decline of regeneration and oxidative defense mechanisms in aging liver.     

Up-regulation of dicer, a component of the MicroRNA machinery, in prostate adenocarcinoma

MicroRNAs are small noncoding 18- to 24-nt RNAs that are predicted to regulate expression of as many as 30% of protein-encoding genes. In prostate adenocarcinoma, 39 microRNAs are up-regulated, and six microRNAs are down-regulated. Production and function of microRNA requires coordinated processing by proteins of the microRNA machinery. Dicer, an RNase III endonuclease, is an essential component of the microRNA machinery. From a gene array analysis of 16 normal prostate tissue samples, 64 organ-confined, and four metastatic prostate adenocarcinomas, we identified an up-regulation of major components of the microRNA machinery, including Dicer, in metastatic prostate adenocarcinoma. Immunohistochemical studies on a tissue microarray consisting of 232 prostate specimens confirmed up-regulation of Dicer in prostatic intraepithelial neoplasia and in 81% of prostate adenocarcinoma. The increased Dicer level in prostate adenocarcinoma correlated with clinical stage, lymph node status, and Gleason score. Western blot analysis of benign and neoplastic prostate cell lines further confirmed Dicer up-regulation in prostate adenocarcinoma. Dicer up-regulation may explain an almost global increase of microRNA expression in prostate adenocarcinoma. The presence of up-regulated microRNA machinery may predict the susceptibility of prostate adenocarcinoma to RNA interference-based therapy.     

Increased fatty acid uptake, a factor in increased hepatic triacylglycerol synthesis in aging rats

The mechanisms responsible for the increased hepatic triacylglycerol synthesis in aging are not established. We studied [1-14C] palmitate uptake and its esterification to triacylglycerols in the isolated hepatocytes of 2-month, 10-month and 20-month-old normal rats. In all hepatocytes, palmitate uptake and its esterification were linearly related to medium palmitate concentration, but palmitate uptake and triacylglycerol synthesis by the hepatocytes of 10-month and 20-month-old rats were nearly double that observed with the cells of 2-month-old rats. These results suggest that increased fatty acid uptake by the liver cells was a contributory factor in the increased triacylglycerol synthesis observed in the liver of senescent rats. The changes in the hepatocyte leading to increased fatty acid uptake and hence increased triacylglycerol synthesis are detected as early as middle age of the rat.     

Changes in the levels and the rate of synthesis of transfer RNA in tissues of mice of different ages

Levels of transfer RNA (tRNA) were determined in liver, kidney, skeletal muscle, heart, and brain of young (35-day), adult (12-month) and old (24-month) female C57BL/6J mice. Kidney and liver showed little change in tRNA levels between young and adult mice, but the levels decreased in old mice. Skeletal muscle tRNA decreased steadily from young to old mice. Heart tRNA increased during maturation to adult organisms and then decreased in old individuals. Brain levels of tRNA increased steadily.No age-related change in the rate of transport of orotic acid into cells was observed. However, all tissues exhibited a decrease in uridine pools between adult and old mice. Most importantly, all tissues of aging mice showed a decrease in the rate of tRNA synthesis.     

Age-related spatial cognitive impairment is correlated with a decrease in ChAT in the cerebral cortex,hippocampus and forebrain of SAMP8 mice

At present, the mechanisms underlying cognitive disorders remain unclear. The senescence-accelerated mice (SAM) prone/8 (P8) has been proposed as a useful model for the study of aging, and SAM resistant/1 (R1) is its control as a normal aging strain. The purpose of this study was to investigate choline acetyltransferase (ChAT) expression in SAM brain. The age-related decline of learning and memory ability in P8 mice (4, 8 and 12 months old, n = 10 for each group) was proved in Morris water maze test (MWM). After the behavioral test, protein and mRNA levels of ChAT were determined in the cerebral cortex, hippocampus and forebrain by means of immunostaining, Western blotting, and real time quantitative PCR (QPCR). Comparing with 4-month-old P8 and R1, 8- and 12-month-old P8 showed age-related cognitive impairment in MWM test. The latencies of the 4-month-old P8 in a hidden platform trial were significantly shorter, and the retention time was significantly longer than that of the older P8 groups. In addition, significantly low level of ChAT protein was observed in older P8 groups. Comparing with the 4-month-old P8, ChAT mRNA in the 12-month-old P8 declined significantly in all three regions of P8 brain. Pearson correlation test showed that the latencies in the MWM were positively correlated with the level of ChAT in P8. Such phenomenon could not be detected in normal aging R1 mice. These findings suggest that the decrease of ChAT in P8 mice was responsible for the age-related learning and memory impairments in some sense.     

Age-related changes in activation of telomerase in the bone marrow of normal and thymectomized mice

Age-related and thymus-dependent regulation of telomerase activity was studied in the bone marrow of normal (physiological aging) and thymectomized (experimental aging) mice. There was no strong correlation between the age and telomerase activity in bone marrow cells of normal mice. We observed only small individual differences in telomerase activity. Thymectomy 2-fold increased telomerase expression in young (2-month-old) and old (24-month-old) animals. Individual differences in telomerase activity in the bone marrow of thymectomized mice were more pronounced and did not depend on the age. The role of the thymus in cell aging is discussed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 6, pp. 654–656, June, 2000     

Decreased rates of protein synthesis by cell-free preparations from different organs of aging mice

Protein synthetic activity was determined in postmitochondrial preparations from heart, brain, kidney, liver and skeletal muscle of 5-26-month-old female C57B1/6J mice. An age-dependent decrease in the rate of protein synthesis was exhibited by all preparations except heart muscle. A 65% decrease in translational rate was found in liver, with the greatest decrease appearing after 21 months. Translation in the brain preparation declined little during the first 20 months, but dropped 33% between 20 and 26 months. The kidney preparation decreased 30% during the first 16 months and 70% by the end of 26 months of age. Skeletal muscle showed an overall decrease of 85% in translation rate. In contrast, heart muscle decreased no more than 10% over the life-span of the mice. From these results, it appears that aging has a differential effect on protein synthesis in different kinds of cells.     

microRNA与心肌缺血损伤研究新进展

背景：研究发现多种microRNA在心肌缺血缺氧时表达明显异常,它们对心血管疾病的发生和发展起着重要作用。 目的：介绍近5年来microRNA对心肌缺血损伤的影响、作用机制及可能的治疗方案。 方法：分别以“microRNA、心肌、缺血、缺氧”为检索词,应用计算机检索PubMed 数据库和ISI Web of Knowledge平台检索近5年有关文章,排除与研究目的无关和内容重复者,保留42篇文献做进一步分析。 结果与结论：microRNA是一类具有转录后调节活性的内源性小分子RNA,通过与靶mRNA的3`端非编码区结合负性调控基因的表达而参与心血管疾病的发生发展。目前研究表明microRNA参与了心肌缺血、缺血后心脏重塑、心肌梗死后继发性心律失常等相关的病理过程,人工干预microRNA的表达可以加剧或预防心肌缺血缺氧损伤的进展。microRNA可能成为治疗心血管疾病的靶向分子。     

Caloric restriction and aging but not overexpression of SOD1 affect hippocampal volumes in mice

Caloric restriction (CR) and antioxidants have been proposed as strategies to attenuate age-related brain changes. The hippocampus and its subregions dentate gyrus (DG), CA3 and CA1-2 show vulnerability to aging, with hippocampal volume alterations as a measurable sign. Using design-based stereological techniques, we investigated the volumes of the hippocampus and its subregions in six 12-month-old and six 24-month-old mice that were randomly selected from four aging cohorts of 60 male mice each: (1) wild-type mice (WT) fed with control diet (CD), (2) transgenic mice oxerexpressing normal human SOD1 fed with CD, (3) WT mice fed with CR diet, and (4) SOD1 mice fed with CR diet. Aging reduced the mean volume of the entire hippocampus (−9.5%), grey (−8.7%) and white matter (−9.7%), and CA3 subregion (−13.6%), but not DG or CA1-2 subregion. CR reduced the mean volumes of every hippocampal region investigated (on average −11%) in both 12-month-old, and 24-month-old mice. Overexpression of SOD1 was not associated with any volume alteration. These findings indicate that although aging and CR in mice are both associated with hippocampal volume reductions, the patterns of the volume reductions differ. These morphometric alterations may have impact on the function of the hippocampus during aging and CR.     

Age and exercise-related changes in myocardial mitochondria in mice

A quantitative histochemical and an ultrastructural study was performed on the left ventricular myocardium of C57BL/6J female mice to evaluate the effects of aging and running exercise on mitochondrial structure and SDH activity. A comparison was made between 6-month-old ("young") and 27-month-old ("old") sedentary mice with age-matched mice subjected to running schedules (10 or 30 min/d) for 6 weeks. In longer-term studies, 27-month-old mice were given similar daily running schedules over a 10 month period ("long term runners"). No significant differences were found in SDH activity during the normal course of aging in mice (6 to 27 months of age), however, the response to running differed markedly in young versus old mice. "Young" trained mice showed significant increase in SDH activity compared with age-matched sedentary mice, whereas "old" trained mice showed significantly reduced SDH activity. Electron microscopy showed ultrastructural changes in mitochondria associated with aging including the development of large aggregations of mitochondria in subsarcolemmal and paranuclear sites. Running schedules, especially in aged runners, caused an increase in interfibrillar mitochondria hypertrophy, loss of matrix and cristae, incorporation of lipid inclusions and the formation of giant mitochondria. These abnormal mitochondrial changes are interpreted as being degenerative and possibly contributing to the reduced SDH levels found in cardiac myocytes of aged runners. Our results indicate that whereas regular exercise in young animals enhanced SDH activity, in aged mice it may be detrimental rather than beneficial.     

Reference memory,anxiety and estrous cyclicity in C57BL/6NIA mice are affected by age and sex

Age-related changes in learning and memory are common in rodents. However, direct comparisons of the effects of aging on learning and memory in both males and females are lacking. The present study examined whether memory deteriorates with increasing age in C57BL/6NIA mice, and whether age-related changes in learning and memory are similar in both sexes. Male and female mice (five, 17 and 25 months of age) were tested in a battery of behavioral tasks including the Morris water maze (spatial and non-spatial reference memory), simple odor discrimination (olfactory reference memory), plus maze (anxiety/exploration), locomotor activity, and basic reflexes. Five-month-old mice learned the water maze and odor discrimination tasks rapidly. Relative to five-month-old mice, 25-month-old mice exhibited impaired spatial and olfactory reference memory, but intact non-spatial reference memory. The spatial reference memory of 17-month-old mice was also impaired, but less so than 25-month mice. Seventeen-month-old mice exhibited intact non-spatial (visual and olfactory) reference memory. Five and 25-month-old mice had similar levels of plus maze exploration and locomotor activity, whereas 17-month-old mice were more active than both groups and were slightly less exploratory than five-month-old mice. Although sex differences were not observed in the five- and 25-month groups, 17-month-old females exhibited more impaired spatial reference memory and increased anxiety relative to 17-month-old males. Estrous cycling in females deteriorated significantly with increased age; all 25-month-old females had ceased cycling and 80% of 17-month-old females displayed either irregular or absent estrous cycling.This study is the first to directly compare age-related mnemonic decline in male and female mice. The results suggest that: (i) aged mice exhibit significant deficits in spatial and olfactory reference memory relative to young mice, whereas middle-aged mice exhibit only a moderate spatial memory deficit and; (ii) spatial reference memory decline begins at an earlier age in females than in males, a finding that may be related to the cessation of estrous cycling.     

Reference memory, anxiety and estrous cyclicity in C57BL/6NIA mice are affected by age and sex

Age-related changes in learning and memory are common in rodents. However, direct comparisons of the effects of aging on learning and memory in both males and females are lacking. The present study examined whether memory deteriorates with increasing age in C57BL/6NIA mice, and whether age-related changes in learning and memory are similar in both sexes. Male and female mice (five, 17 and 25 months of age) were tested in a battery of behavioral tasks including the Morris water maze (spatial and non-spatial reference memory), simple odor discrimination (olfactory reference memory), plus maze (anxiety/exploration), locomotor activity, and basic reflexes. Five-month-old mice learned the water maze and odor discrimination tasks rapidly. Relative to five-month-old mice, 25-month-old mice exhibited impaired spatial and olfactory reference memory, but intact non-spatial reference memory. The spatial reference memory of 17-month-old mice was also impaired, but less so than 25-month mice. Seventeen-month-old mice exhibited intact non-spatial (visual and olfactory) reference memory. Five and 25-month-old mice had similar levels of plus maze exploration and locomotor activity, whereas 17-month-old mice were more active than both groups and were slightly less exploratory than five-month-old mice. Although sex differences were not observed in the five- and 25-month groups, 17-month-old females exhibited more impaired spatial reference memory and increased anxiety relative to 17-month-old males. Estrous cycling in females deteriorated significantly with increased age; all 25-month-old females had ceased cycling and 80% of 17-month-old females displayed either irregular or absent estrous cycling. This study is the first to directly compare age-related mnemonic decline in male and female mice. The results suggest that: (i) aged mice exhibit significant deficits in spatial and olfactory reference memory relative to young mice, whereas middle-aged mice exhibit only a moderate spatial memory deficit and; (ii) spatial reference memory decline begins at an earlier age in females than in males, a finding that may be related to the cessation of estrous cycling.     

Transfer of signs of aging to young mice by splenic lymphoid cells from old syngeneic donors

Single intraperitoneal injection of splenic lymphoid cells from 20-month-old mice to 2-month-old syngeneic mice (similarly as 5-day injections of purified brain extract) leads to preterm (4 months earlier than in the control) appearance of aging factor in the blood (the main sign of old age). Combined injections of brain extract and splenic lymphoid cells led to the appearance of aging factor in the blood 5 months earlier than in the control. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 7, pp. 100–102, July, 2007     

Multiple co-localizations in arcuate GHRH-eGFP neurons in the mouse hypothalamus

In the present work, we took advantage of a recently described model of GHRH-enhanced green fluorescent protein (eGFP) transgenic mice to evaluate the extent of co-localization of GHRH neurons with galanin (GAL), neurotensin (NT) and tyrosine hydroxylase (TH) in 3- and 8-month-old male and female mice. The total number of GHRH-eGFP neurons along the rostro-caudal axis of the arcuate nucleus did not differ according to gender or age. GAL-immunoreactivity was present in 40-44% of 3-month-old GHRH-eGFP neurons in male and female arcuate nucleus, respectively, but only 25-22% in 8-month-old mice. TH immunoreactivity occurred in 36-35% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice and these proportions increased to 40 and 45% in 8-month-old mice. NT immunoreactivity was present in 14 and 24% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice up to 28 and 26% in 8-month-old mice. Thus, co-localization of peptides and enzyme in GHRH-eGFP neurons displays a sexual dimorphism at 3-month of age for NT, and at 8-month for TH, while the total number of GHRH-eGFP neurons does not exhibit gender difference at either age. In summary, it appears that changes in co-localized (and presumably co-released) peptides, rather than GHRH per se, may contribute to the changes in sexually dimorphic GH secretion with aging in the mouse.     

Role of microRNA-15a in autoantibody production in interferon-augmented murine model of lupus

MicroRNAs (miRNAs) are involved in the regulation of immunity via targeting of mRNA encoding immune response elements. In this report, alterations in the expression of microRNAs as autoantibody levels increase was investigated. The (NZB×NZW)F1 or B/W mouse model of systemic lupus erythematosus (SLE) naturally has increased autoantibodies with aging. IFNα (type I IFN) accelerates B/W disease, however, the effects of a related IFN, IFNλ, which is a type III IFN, is largely unknown. The purpose of the study was to investigate the relationship between IFN-accelerated disease, microRNAs, immunoregulatory B cell subsets and autoantibody production in the autoimmune-prone environment in vivo. B/W mice received osmotic pumps to chronically deliver IFNα and IFNλ for up to 16 weeks. Urine protein level was monitored weekly by urine strips and proteinuria was used as the disease marker. Splenic cells were taken for flow analysis of B cell subsets and levels of microRNAs determined. Plasma were analyzed for autoantibodies and microRNA levels. As a result of treatment, IFNα accelerated proteinuria in a dose dependent manner, while IFNλ single treatment did not show a significant effect, but greatly enhanced low dose IFNα effects in the combination treatment. Both the splenic cellular and plasma miR-15a were elevated in diseased compared to pre-diseased mice as well as autoantibody levels. Autoantibodies and miR-15a levels were significantly correlated. The immunosuppressive B subpopulation, B-10, was reduced following IFNα treatment. In addition in diseased mice, B-10 versus B-2 ratios were reduced in IFN-treated B/W compared to the control PBS treated group. In all B/W the miR-15a was highly expressed in the B-10 subset and this increased with disease development, suggesting that miR-15a has a specific negative effect on the B-10 subpopulation. In conclusion, our data support the involvement of elevated miR-15a in autoimmune disease development in B/W mice and suggest that decreasing this microRNA might be beneficial in B/W mice.     

Changes of 8-hydroxydeoxyguanosine levels in rat organ DNA during the aging process.

We investigated the accumulation of oxidative DNA damage during the aging process by using 8-hydroxydeoxyguanosine (8-OH-dG) as a marker. The 8-OH-dG is one of the oxidative DNA damage products and is supposed to be a critical factor in carcinogenesis involving oxygen radicals. The 8-OH-dG levels in the DNA of liver, kidney, brain, lung, and spleen were measured in male and female F344 rats 6- to 30-month-old. The 8-OH-dG levels in the liver and kidney DNA of male rats increased significantly with age, but did not change in brain, lung, and spleen. Similarly, the 8-OH-dG levels in the liver and kidney DNA of female rats significantly increased with age, while changes in the brain, lung, and spleen DNA were much smaller. These results indicate that the accumulation of oxidative DNA damage during the aging process varies among organs, with slight sex difference.     

Age-related changes in antioxidant enzyme activities are region and organ, as well as sex, selective in the rat.

Enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were determined in the liver as well as several specific brain regions of young and old Fischer-344 rats of both sexes. In the liver of male rats, activities of CAT as well as Mn-SOD were lower, while activities of Cu Zn-SOD were higher in old (30-month-old) rats than in young (7-month-old) ones. Activities of total SOD as well as GSH Px were comparable for young and old male rat livers. In contrast to male rats, in female rat livers, activities of CAT were significantly higher in old (28-months-old) rats, while activities of Mn-SOD were slightly (but significantly) higher in old rat livers. In old male rats, activities of Mn-SOD were significantly higher than in young males in several specific regions of the brain (the substantia nigra (s. nigra), striatum, hippocampus) but lower in the cerebellum. In particular, SOD activities in s. nigra, striatum and hippocampus in old male rats were several fold higher than corresponding values in young male rats. Activities of Cu Zn-SOD were generally unchanged with age. Activities of CAT as well as GSH-Px (both Se-dependent and non-Se-dependent forms) were also relatively unaffected by age. In female rat brains, activities of Mn-SOD as well as those of others all remained mostly unaffected by aging, although there was a general tendency of slightly higher activities in most cerebral regions for Mn-SOD in old female rats. Thus, age-related changes of these antioxidant enzymes in the liver and brain are markedly sex dependent and some enzyme activities (such as CAT in the liver) change in an opposite direction with age. Changes of Mn-SOD in the brain were markedly region-specific in male rats. Results suggest that the significance of the changes of these antioxidant enzyme activities during aging needs to be carefully interpreted, taking into consideration the fact that changes are markedly variable depending on sex as well as the organs and brain regions examined.     

MicroRNA profiling of cystic fibrosis intestinal disease in mice

Cystic fibrosis (CF) intestinal disease is characterized by alterations in processes such as proliferation and apoptosis which are known to be regulated in part by microRNAs. Herein, we completed microRNA expression profiling of the intestinal tissue from the cystic fibrosis mouse model of cystic fibrosis transmembrane conductance regulator (Cftr) deficient mice (BALBc/J Cftr(tm1UNC)), relative to that of wildtype littermates, to determine whether changes in microRNA expression level are part of this phenotype. We identified 24 microRNAs to be significantly differentially expressed in tissue from CF mice compared to wildtype, with the higher expression in tissue from CF mice. These data were confirmed with real time PCR measurements. A comparison of the list of genes previously reported to have decreased expression in the BALB×C57BL/6J F2 CF intestine to that of genes putatively targeted by the 24 microRNAs, determined from target prediction software, revealed 155 of the 759 genes of the expression profile (20.4%) to overlap with predicted targets, which is significantly more than the 100 genes expected by chance (p=1×10(-8)). Pathway analysis identified these common genes to function in phosphatase and tensin homolog-, protein kinase A-, phosphoinositide-3 kinase/Akt- and peroxisome proliferator-activated receptor alpha/retinoid X receptor alpha signaling pathways, among others, and through real time PCR experiments genes of these pathways were demonstrated to have lower expression in the BALB CF intestine. We conclude that altered microRNA expression is a feature which putatively influences both metabolic abnormalities and the altered tissue homeostasis component of CF intestinal disease.