Down regulation of neuronatin by microRNA-151 overcomes inhibition of axonal growth by myelin-based inhibitors
Item
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Title
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Down regulation of neuronatin by microRNA-151 overcomes inhibition of axonal growth by myelin-based inhibitors
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Identifier
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d_2009_2013:4b870df92200:10418
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identifier
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10491
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Creator
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Kochanek, Dawn Marie,
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Contributor
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Marie T. Filbin
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Date
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2010
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Language
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English
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Publisher
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City University of New York.
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Subject
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Molecular biology | Neurosciences | axonal regeneration | cAMP | microRNA | Neuronatin
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Abstract
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After injury, the axons of the adult central nervous system (CNS) fail to regenerate. This failure is due to the cellular environment and the neuronal response to that environment. One factor for environmentally-mediated axonal inhibition are the proteins that are present in myelin, such as myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein (OMgp). Previously, our lab has shown that elevating the ubiquitous second messenger cyclic-adenosine monophosphate (cAMP) overcomes MAG/myelin inhibition. MicroRNAs (miRNA or miR), are small fragments of RNA that have been shown to bind to target mRNAs and regulate their translation. We hypothesized that miRNAs might be playing a role in the ability of cAMP to overcome MAG/myelin-based axonal growth inhibition. To investigate if miRNAs have a role in the cAMP effect we performed a miRNA microarray with cAMP treated vs. control dorsal root ganglion (DRG) neurons. One miRNA that increased more than two fold with cAMP treatment was miRNA-151. As previously stated, the axons of the mammalian CNS do not regenerate after injury. However, there is one situation in which they have been shown to spontaneously regenerate. DRG axons are bifurcated with one branch extending into the CNS and the other into the peripheral nervous system (PNS). Studies have shown that if a lesion is made to the PNS branch and then subsequently to the CNS branch, the CNS branch will regenerate. This "conditioning lesion"-induced regeneration has been found to be dependent upon increased levels of cAMP. We next investigated whether like after treatment with cAMP, miR-151 was also increased after a peripheral conditioning lesion. We found similar significant increases in miR-151 levels in DRG neurons following a peripheral conditioning lesion. To determine a functional role for miR-151 in overcoming MAG/myelin-mediated neurite outgrowth inhibition we next performed overexpression and knockdown analyses of miR-151 and then subsequently subjected the neurons to a neurite outgrowth assay. Overexpression of pre-miR-151 in DRG neurons overcame MAG/myelin-mediated neurite outgrowth inhibition and conversely, knockdown of miR-151 with anti-miR-151 in DRG neurons attenuated the ability of db-cAMP to overcome MAG/myelin-mediated inhibition. To investigate the mechanism by which overexpression of miR-151 overcomes MAG/myelin-mediated inhibition we sought to identify miR-151 target mRNAs, using target prediction algorithms. One putative target was Neuronatin, a 9 kD transmembrane proteolipid protein with unknown neuronal function. We next wanted to assess if miR-151 could bind to the 3'UTR of Neuronatin and inhibit its translation. To test this, 293-T cells were co-transfected with miR-151 and a luciferase reporter gene fused to a wildtype or mutated Neuronatin 3'UTR. MiR-151 overexpression decreased the luciferase activity of the wildtype, but not the activity of the mutated Neuronatin 3'UTR, thus validating that Neuronatin is a miR-151 target. Likewise, we found that both treatment of DRG neurons with db-cAMP or overexpressing miR-151 led to a significant decrease in Neuronatin protein levels, while Neuronatin mRNA levels were unaffected. Finally, using siRNA we knocked-down Neuronatin in DRG neurons and then subjected the neurons to a neurite outgrowth assay. Knockdown of Neuronatin led to a significant increase in total neurite length on both MAG-expressing CHO cells and purified myelin. Our findings suggest that the cAMP-induced miR-151 plays an important role in overcoming MAG/myelinmediated axonal growth inhibition.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Biology
