NMDA receptor-dependent regulation of miRNA expression and association with Argonaute during LTP in vivo.

microRNAs (miRNAs) are major regulators of protein synthesis in the brain. A major goal is to identify changes in miRNA expression underlying protein synthesis-dependent forms of synaptic plasticity such as long-term potentiation (LTP). Previous analyses focused on changes in miRNA levels in total lysate samples. Here, we asked whether changes in total miRNA accurately reflect changes in the amount of miRNA bound to Argonaute protein within the miRNA-induced silencing complex (miRISC). Ago2 immunoprecipitation was used to isolate RISC-associated miRNAs following high-frequency stimulation (HFS)-induced LTP in the dentate gyrus of anesthetized rats. Using locked-nucleic acid-based PCR cards for high-throughput screening and independent validation by quantitative TaqMan RT-PCR, we identified differential regulation of Ago2-associated and total miRNA expression. The ratio of Ago2/total miRNA expression was regulated bidirectionally in a miRNA-specific manner and was largely dependent on N-methyl-D-aspartate receptor (NMDA) activation during LTP induction. The present results identify miRNA association with Ago2 as a potential control point in activity-dependent synaptic plasticity in the adult brain. Finally, novel computational analysis for targets of the Ago2-associated miRNAs identifies 21 pathways that are enriched and differentially targeted by the miRNAs including axon guidance, mTOR, MAPK, Ras, and LTP.

MicroRNA regulation of the synaptic plasticity-related gene Arc.

Expression of activity-regulated cytoskeleton associated protein (Arc) is crucial for diverse types of experience-dependent synaptic plasticity and long-term memory in mammals. However, the mechanisms governing Arc-specific translation are little understood. Here, we asked whether Arc translation is regulated by microRNAs. Bioinformatic analysis predicted numerous candidate miRNA binding sites within the Arc 3'-untranslated region (UTR). Transfection of the corresponding microRNAs in human embryonic kidney cells inhibited expression of an Arc 3'UTR luciferase reporter from between 10 to 70% across 16 microRNAs tested. Point mutation and deletion of the microRNA-binding seed-region for miR-34a, miR-326, and miR-19a partially or fully rescued reporter expression. In addition, expression of specific microRNA pairs synergistically modulated Arc reporter expression. In primary rat hippocampal neuronal cultures, ectopic expression of miR-34a, miR-193a, or miR-326, downregulated endogenous Arc protein expression in response to BDNF treatment. Conversely, treatment of neurons with cell-penetrating, peptide nucleic acid (PNA) inhibitors of miR-326 enhanced Arc mRNA expression. BDNF dramatically upregulated neuronal expression of Arc mRNA and miR-132, a known BDNF-induced miRNA, without affecting expression of Arc-targeting miRNAs. Developmentally, miR-132 was upregulated at day 10 in vitro whereas Arc-targeting miRNAs were downregulated. In the adult brain, LTP induction in the dentate gyrus triggered massive upregulation of Arc and upregulation of miR-132 without affecting levels of mature Arc-targeting miRNAs. Turning to examine miRNA localization, qPCR analysis of dentate gyrus synaptoneurosome and total lysates fractions demonstrated synaptic enrichment relative to small nucleolar RNA. In conclusion, we find that Arc is regulated by multiple miRNAs and modulated by specific miRNA pairs in vitro. Furthermore, we show that, in contrast to miR-132, steady state levels of Arc-targeting miRNAs do not change in response to activity-dependent expression of Arc in hippocampal neurons in vitro or during LTP in vivo.

Social Defeat during Adolescence and Adulthood Differentially Induce BDNF-Regulated Immediate Early Genes.

Stressful life events generally enhance the vulnerability for the development of human psychopathologies such as anxiety disorders and depression. The incidence rates of adult mental disorders steeply rises during adolescence in parallel with a structural and functional reorganization of the neural circuitry underlying stress reactivity. However, the mechanisms underlying susceptibility to stress and manifestation of mental disorders during adolescence are little understood. We hypothesized that heightened sensitivity to stress during adolescence reflects age-dependent differences in the expression of activity-dependent genes involved in synaptic plasticity. Therefore, we compared the effect of social stress during adolescence with social stress in adulthood on the expression of a panel of genes linked to induction of long-term potentiation (LTP) and brain-derived neurotrophic factor (BDNF) signaling. We show that social defeat during adolescence and adulthood differentially regulates expression of the immediate early genes BDNF, Arc, Carp, and Tieg1, as measured by qPCR in tissue lysates from prefrontal cortex, nucleus accumbens, and hippocampus. In the hippocampus, mRNA levels for all four genes were robustly elevated following social defeat in adolescence, whereas none were induced by defeat in adulthood. The relationship to coping style was also examined using adult reactive and proactive coping rats. Gene expression levels of reactive and proactive animals were similar in the prefrontal cortex and hippocampus. However, a trend toward a differential expression of BDNF and Arc mRNA in the nucleus accumbens was detected. BDNF mRNA was increased in the nucleus accumbens of proactive defeated animals, whereas the expression level in reactive defeated animals was comparable to control animals. The results demonstrate striking differences in immediate early gene expression in response to social defeat in adolescent and adult rats.

Analysis of three phenotyping methods and pulsed-field gel electrophoresis for differentiation of methicillin resistant Staphylococcus aureus isolated from two hospitals in Thailand.

Methicillin resistant Staphylococcus aureus (MRSA) is an important hospital and community-acquired pathogen. Rapid and reliable epidemiologic typing is necessary for controlling the spread of MRSA outbreak. The objective of this study was to compare the phenotyping with the genotyping method to differentiate MRSA isolates obtained from the two hospitals in Thailand (central and northeastern). Seventy-four MRSA isolates were randomly collected and confirmed by the presence of mecA gene. Antibiogram, phage typing and enterotoxin production were used for the phenotyping analysis. Pulsed-field gel electrophoresis (PFGE) with Smal digestion of chromosomal DNA was used for the genotyping analysis. We found 17 distinct profiles by the 3 phenotypic typing methods and 18 PFGE types designated as 5 major types (A-E) and 13 subtypes. The most frequent PFGE types and their related subtypes found in both hospitals were A and C, comprising 54 and 27%, respectively. The antibiogram could differentiate 6 different types. All isolates were resistant to the majority of antimicrobial agents tested, but were susceptible to vancomycin and fosfomycin. Ten (13.5%) MRSA isolates produced enterotoxin A. Nontypable phage and phage type 77 were found predominantly in MRSA isolated from the northeast and central hospital, respectively. A significant correlation was found between the phenotyping and the genotyping methods and there was a good correlation between antibiogram and PFGE. Antibiogram typing alone can be used as a useful epidemiological marker for practical purposes. PFGE types A and C were the common endemic MRSA clones in both hospitals in Thailand.

Evaluation of polymerase chain reaction, conventional and MRSA screen latex agglutination methods for detection of methicillin-resistant, -borderline and -susceptible Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), is difficult and expensive to treat, therefore early screening is essential. Several phenotypic and genotypic methods are used to detect MRSA; however, the method of choice remains problematic. We have evaluated four phenotypic methods, broth microdilution (MIC), oxacillin disk agar diffusion (ODD), oxacillin screening salt agar (OSS), and a new rapid phenotypic (MRSA screen latex agglutination, MSLA) with the genotypic gold standard of PCR mecA detection to determine the most appropriate method for routine laboratory use. We randomly collected 203 S. aureus isolates from patients and carriers at two hospitals in Thailand. Using MIC method, three sub-groups were differentiated from among these isolates, namely MRSA (106 isolates), borderline-resistant S. aureus (BRSA) (65 isolates), and methicillin-susceptible S. aureus (MSSA)(32 isolates). A total of 10 methicillin-resistant S. epidermidis (MRSE) isolates were also included. The sensitivity and specificity of MIC, ODD, OSS, and MSLA were 99 and 96, 100 and 97, 100 and 97, and 100 and 100%, respectively. Our study indicated that ODD is still appropriate for routine laboratory. MSLA had the highest sensitivity and specificity and is rapid but expensive, so is the most appropriate method for emergency cases. MIC method was better for BRSA detection and OSS method was more appropriate for screening clinical specimens and carriers.

Evaluation of different primers for detecting mecA gene by PCR in comparison with phenotypic methods for discrimination of methicillin-resistant Staphylococcus aureus.

Detection of the mecA gene by polymerase chain reaction (PCR) is the gold standard for identifying methicillin-resistant Staphylococcus aureus (MRSA). PCR assays, employing MR1-MR2 primers (primer set 1) and MR3-MR4 primers (primer set 2) to generate 154 and 533 bp fragment, respectively, are most widely used for amplification of mecA gene. The purpose of this study was to evaluate the presence of mecA gene in 100 clinical isolates of S. aureus using PCR with the two pairs of primers. The results were compared to the broth dilution MIC method, oxacillin salt screening method (OSS) and oxacillin disk agar diffusion method (ODD). Fifteen of the 100 isolates showed a discrepancy between the mecA primer sets 1 and 2. Three isolates (3%) without the mecA gene showed discrepancies with phenotypic methods. The sensitivity, specificity and positive and negative predictive values for the 154 and 533 bp products of mecA were 79, 85, 83, 81 and 94, 100, 100, 94%, respectively. The results indicated that primer set 2 was more appropriate than primer set 1 for the detection of mecA gene in MRSA. There was a good correlation among the mecA gene detection, ODD and OSS methods. The discrepancy of three isolates between PCR and phenotypic methods should be clarified for other resistant mechanisms.