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MicroRNAs (miRNAs) regulate cellular fate by controlling the stability or translation of mRNA transcripts. Although the spatial and temporal patterning of miRNA expression is tightly controlled, little is known about signals that induce their expression nor mechanisms of their transcriptional regulation. Furthermore, few miRNA targets have been validated experimentally. The miRNA, miR132, was identified through a genome-wide screen as a target of the transcription factor, cAMP-response element binding protein (CREB). miR132 is enriched in neurons and, like many neuronal CREB targets, is highly induced by neurotrophins. Expression of miR132 in cortical neurons induced neurite outgrowth. Conversely, inhibition of miR132 function attenuated neuronal outgrowth. We provide evidence that miR132 regulates neuronal morphogenesis by decreasing levels of the GTPase-activating protein, p250GAP. These data reveal that a CREB-regulated miRNA regulates neuronal morphogenesis by responding to extrinsic trophic cues.

Activity-regulated gene expression is believed to play a key role in the development and refinement of neuronal circuitry. Nevertheless, the transcriptional networks that regulate synapse growth and plasticity remain largely uncharacterized. Here, we show that microRNA 132 (miR132) is an activity-dependent rapid response gene regulated by the cAMP response element-binding (CREB) protein pathway. Introduction of miR132 into hippocampal neurons enhanced dendrite morphogenesis whereas inhibition of miR132 by 2′ O -methyl RNA antagonists blocked these effects. Furthermore, neuronal activity inhibited translation of p250GAP, a miR132 target, and siRNA-mediated knockdown of p250GAP mimicked miR132-induced dendrite growth. Experiments using dominant-interfering mutants suggested that Rac signaling is downstream of miR132 and p250GAP. We propose that the miR132–p250GAP pathway plays a key role in activity-dependent structural and functional plasticity.

Newborn neurons in the dentate gyrus of the adult hippocampus rely upon cAMP response element binding protein (CREB) signaling for their differentiation into mature granule cells and their integration into the dentate network. Among its many targets, the transcription factor CREB activates expression of a gene locus that produces two microRNAs, miR-132 and miR-212. In cultured cortical and hippocampal neurons, miR-132 functions downstream from CREB to mediate activity-dependent dendritic growth and spine formation in response to a variety of signaling pathways. To investigate whether miR-132 and/or miR-212 contribute to the maturation of dendrites in newborn neurons in the adult hippocampus, we inserted LoxP sites surrounding the miR-212/132 locus and specifically targeted its deletion by stereotactically injecting a retrovirus expressing Cre recombinase. Deletion of the miR-212/132 locus caused a dramatic decrease in dendrite length, arborization, and spine density. The miR-212/132 locus may express up to four distinct microRNAs, miR-132 and -212 and their reverse strands miR-132* and -212*. Using ratiometric microRNA sensors, we determined that miR-132 is the predominantly active product in hippocampal neurons. We conclude that miR-132 is required for normal dendrite maturation in newborn neurons in the adult hippocampus and suggest that this microRNA also may participate in other examples of CREB-mediated signaling.

Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor originally identified through its ability to interact with adenovirus E1A. The finding that CtBP–E1A interactions were regulated by the nicotinamide adeninine dinucleotides NAD ⁺ and NADH raised the possibility that CtBP could serve as a nuclear redox sensor. This model requires differential binding affinities of NAD ⁺ and NADH, which has been controversial. The structure of CtBP determined by x-ray diffraction revealed a tryptophan residue adjacent to the proposed nicotinamide adenine dinucleotide binding site. We find that this tryptophan residue shows strong fluorescence resonance energy transfer to bound NADH. In this report, we take advantage of these findings to measure the dissociation constants for CtBP with NADH and NAD ⁺ . The affinity of NADH was determined by using fluorescence resonance energy transfer. The binding of NADH to protein is associated with an enhanced intensity of NADH fluorescence and a blue shift in its maximum. NAD ⁺ affinity was estimated by measuring the loss of the fluorescence blue shift as NADH dissociates on addition of NAD ⁺ . Our studies show a >100-fold higher affinity of NADH than NAD ⁺ , consistent with the proposed function of CtBP as a nuclear redox sensor. Moreover, the concentrations of NADH and NAD ⁺ required for half-maximal binding are approximately the same as their concentrations in the nuclear compartment. These findings support the possibility that changes in nuclear nicotinamide adenine dinucleotides could regulate the functions of CtBP in cell differentiation, development, or transformation.