One distinguishing attribute of the mammalian hippocampus is its ability to continuously produce newborn neurons throughout adult life. These newborn neurons are first exposed to GABAergic signals as they emerge from the subgranule layer of the dentate gyrus. This activity drives a CREB-dependent signaling pathway that alters the gene expression within that newborn neuron, and as such, is required for its maturation and survival. Nevertheless, the exact mechanism is still poorly understood as to how CREB-signaling translates into the maturation, survival, and functional integration of that particular cell into the hippocampal circuitry. We do know that one of the important CREB targets for the maturation of adult newborn neurons in vivo is non-coding transcript microRNA-132 (miR-132). When we excised the miR-132 locus in adult newborn neurons it resulted in an immature dendritic arbor that phenocopied disruption of CREB signaling. Nevertheless as a microRNA, miR-132 has the ability to downregulate a number of diverse transcripts. The important issue that this proposal addresses is the identification and characterization of specific miR-132 targets that contribute to this establishment of the dendritic arbor. Without understanding the identity of specific cellular targets, we cannot fully appreciate the regulation conferred by miR-132, and only know that its activity is required for this process. Through preliminary screens and the availability of a mouse model, I am in a unique position to characterize how the regulation of candidate target Heparin-binding Epithelial Growth Factor (Hb-EGF) by miR- 132 contributes to the dendritic arbor of newborn neurons. Preliminary data demonstrate that levels of Hb-EGF are dramatically downregulated specifically in response to miR-132 activity.
Aim 1 takes advantage of being able to target excision of the miR-132 locus in the adult newborn neurons of the floxed mouse model to examine how Hb-EGF is regulated by miR-132 in these cells. Changes in Hb-EGF levels will be evaluated for how they affect dendritic arborization, and then we will determine whether depletion of Hb-EGF can rescue the immature dendritic phenotype that occurs with miR-132 activity is ablated in adult newborn neurons. It is likely that other miR-132 direct targets are also important for maturation of the adult newborn neuron. To identify direct miR-132 targets that may function with Hb-EGF, I have developed a molecular miR- 132 target trap that I will develop into a screen in Aim 2. This approach takes advantage of a dominant- negative RNAi-Induced Silencing Complex (RISC)- a central component in the microRNA pathway that bridges the recognition of microRNAs and their targets-to stabilize and capture associated mRNA target transcripts that would otherwise decay via deadenylation and exonuclease activity.
In order to address how adult neurogenesis relates to human aging, disease, and injury, it is absolutely vital to have a clear and detailed understanding of the molecular mechanisms underlying this process. Over the years various cellular factors have been implicated in the establishment and maturation of adult newborn neurons, however, the revelation that activity- dependent microRNA-132 is important in this process provides a unique opportunity to flesh out significant and specific molecular pathways in greater depth. This proposal brings together detailed preliminary data from in vitro molecular studies-which reveal mechanistic insight down to the specific nucleotides that are targeted my miR-132-with important in vivo analysis of how the identified regulation affects the development of these newborn neurons within the context of a whole organism.
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