John Pena

Specificity, duplex degradation and subcellular localization of antagomirs

MicroRNAs (miRNAs) are an abundant class of 20–23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed ‘antagomirs’. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings.

miRNA in situ hybridization in formaldehyde and EDC–fixed tissues

MicroRNAs are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5′ phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid probes by recording nucleic acid melting temperature during ISH.

Differential regulation of mature and precursor microRNA expression by NMDA and metabotropic glutamate receptor activation during LTP in the adult dentate gyrus in vivo

Regulation of microRNA (miRNA) expression and function in the context of activity‐dependent synaptic plasticity in the adult brain is little understood. Here, we examined miRNA expression during long‐term potentiation (LTP) in the dentate gyrus of adult anesthetized rats. Microarray expression profiling identified a subpopulation of regulated mature miRNAs 2 h after the induction of LTP by high‐frequency stimulation (HFS) of the medial perforant pathway. Real‐time polymerase chain reaction analysis confirmed modest upregulation of miR‐132 and miR‐212, and downregulation of miR‐219, while no changes occurred at 10 min post‐HFS. Surprisingly, pharmacological blockade of N‐methyl‐d‐aspartate receptor (NMDAR)‐dependent LTP enhanced expression of these mature miRNAs. This HFS‐evoked expression was abolished by local infusion of the group 1 metabotropic glutamate receptor (mGluR) antagonist, (RS)‐1‐aminoindan‐1,5‐dicarboxylic acid (AIDA). AIDA had no effect on LTP induction or maintenance, but blocked activity‐dependent depotentiation of LTP. Turning to the analysis of miRNA precursors, we show that HFS elicits 50‐fold elevations of primary (pri) and precursor (pre) miR‐132/212 that is transcription dependent and mGluR dependent, but insensitive to NMDAR blockade. Primary miR‐219 expression was unchanged during LTP. In situ hybridization showed upregulation of the pri‐miR‐132/212 cluster restricted to dentate granule cell somata. Thus, HFS induces transcription miR‐132/212 that is mGluR dependent and functionally correlated with depotentiation rather than LTP. In contrast, NMDAR activation selectively downregulates mature miR‐132, ‐212 and ‐219 levels, indicating accelerated decay of these mature miRNAs. This study demonstrates differential regulation of primary and mature miRNA expression by mGluR and NMDAR signaling following LTP induction, the function of which remains to be defined.

Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing

The characterization of post-transcriptional gene regulation by small regulatory (20-30 nt) RNAs, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for characterizing small RNAs is their identification and quantification across different developmental stages, and in normal and disease tissues, as well as model cell lines. Here we present a step-by-step protocol for generating barcoded small RNA cDNA libraries compatible with Illumina HiSeq sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections.

PTEN inhibits adrenomedullin expression and function in brain tumor cells.

SummaryAdrenomedullin is a vasoactive peptide that is upregulated in higher-grade gliomas and promotes tumor cell proliferation. Since reduced activity of the anti-oncogene PTEN seems to also correlate with higher tumor grade, this suggests an inverse association between PTEN activity and adrenomedullin expression. PC12 pheochromocytoma and human U251 glioma cell lines were stably transfected with human PTEN or control plasmid. Adrenomedullin expression was analyzed using quantitative PCR and Western blotting. A cell proliferation assay was used to assess adrenomedullin effects on U251 cells overexpressing PTEN. PC12 and U251 cells overexpressing PTEN had 17- and 8-fold decreases in adrenomedullin mRNA levels, respectively, compared to control cells. Cellular and secreted adrenomedullin peptide was similarly reduced. Addition of adrenomedullin to medium of controlled cells induced proliferation, as described previously, but U251 cells overexpressing PTEN did not respond to exogenous adrenomedullin. Further exploration revealed that PTEN also inhibits expression of the gliomas receptor for adrenomedullin, which accounts for this effect. These data were all replicated with an inducible PTEN construct confirming that these effects are not exclusively secondary to chronic overexpression. Given the profound effects of adrenomedullin on tumor cells, this is a novel and previously unidentified mechanism by which alterations in PTEN levels or function may influence tumor growth.

940. Neuroprotective Effects of XIAP Gene Therapy in Models of Neurodegenerative Diseases

The pathways and mechanisms leading to cell loss in neurodegerative diseases are still largely unknown. Neuronal cell death is thought to occur via apoptosis and the involvement of several caspases at the late stages of this process is well documented. X-linked inhibitor of apoptosis (XIAP) is a potent inhibitor of caspases 9, 3 and 7 and thus represents an attractive candidate as a potentially therapeutic neuroprotective factor. To test this hypothesis, we examined the effects of XIAP in several in vitro models of Parkinsons and Huntingtons disease. Recent reports have documented the pivotal role of the proteasome pathway of protein turnover in the pathogenesis of neurodegenerative disorders and proteasome inhibitors have been employed to model neurodegeneration both in vitro and in vivo. In neuronal cell lines XIAP demonstrated significant protection against apoptosis induced by different proteasome inhibitors. Furthermore, XIAP almost completely prevented cells death induced by overexpression of alpha-synuclein and polyglatamine fusion proteins used to model familial Parkinsons and Huntingtons diseases, respectively. To gain insight into the mechanisms of neuroprotective affects of XIAP we performed analysis of deletion and point mutants of this protein. Our experiments revealed that the N-terminal BIR1 domain as well as the C-terminal RING domain are dispensable for anti-apoptotic effects. Deletion or point mutation of the BIR2 domain or the juxtaposed linker region partially abrogated XIAP activity. In contrast, anti-apoptotic effect was lost when a deletion or a point mutation were introduced into the BIR3 domain. Caspases 3 and 7 are believed to be bound by the BIR2 and the linker regions while the caspase 9 is inactivated by the BIR3 domain. Thus, our data suggest that the protective affects of XIAP in these two models of Parkinsons and Huntingtons diseases are mostly mediated by the anti-caspase 9 activity. To explore neuroprotective effects of XIAP in vivo we have generated a recombinant AAV (rAAV) vector expressing a stabilized truncated from of XIAP lacking the RING domain (dXIAP). Slowly progressing nigral degeneration triggered by proteasome inhibition is believed to be a close recapitulation of the events that mark sporadic Parkinsons disease in humans. Therefore, for this study we employed a novel PSI rat model of Parkinsons disease which is induced by inhibition of proteasome machinery. While treatment of the animals with PSI resulted in approximately 50% loss of dopaminergic neurons in the substantia nigra on the side injected with a control vector, all the cells were protected on the contralateral side injected with the dXIAP-overexpressing rAAV. Thus, dXIAP delivered by a rAAV provides a long-term protection of nigral neurons against death induced by proteasome inhibition. Furthermore, to our knowledge this is the first demonstration that neuronal loss can be prevented in this novel model of Parkinsons disease. Thus, our studies may provide a foundation for the use of dXIAP for neuroprotection, which can be further exploited for human gene therapy.