MicroRNAs shape the neuronal landscape

Abstract

The nervous system equips us with capability to adapt to many conditions and circumstances. We rely on an armamentarium of intricately formed neural circuits for many of our adaptive strategies. However, this capability also depends on a well-conserved toolkit of different molecular mechanisms that offer not only compensatory responses to a changing world, but also provide plasticity to achieve changes in cellular state that underlie a broad range of processes from early developmental transitions to life-long memory. Among the molecular tools that mediate changes in cellular state, our understanding of posttranscriptional regulation of gene expression is expanding rapidly. Part of the "epigenetic landscape" that shapes the deployment and robust regulation of gene networks during the construction and the remodeling of the brain is the microRNA system controlling both levels and translation of messenger RNA. Here we consider recent advances in the study of microRNA-mediated regulation of synaptic form and function.

Figure 1. Spatial and Temporal Domains in Genome Expression and Function

A. Waddington’s adaptive “gun” response triggered by environmental stimuli or information from neirghboring cells can utilize a broad range of molecular mechanisms to mediate changes in the expression of the genome to alter phenotypes or cellular behavior. B. This diagram represents the relative effective spatial and temporal range of function and adaptive response for different mechanisms in the progression from primary nuclear production of mRNA (transcription, splicing, processing and export; in yellow), to mRNA delivery (transport and localization; in orange), to posttranscriptional miRNA regulation of mRNA (mRNA stability and access to translational machinery; in red), to active translation (in purple) and to the final function of the encoded protein(s) (in blue). While transcriptional mechanisms can be sustained for prolonged periods of cellular and/or organismic lifetime, these processes are slow to respond and have very limited spatial acuity. Posttranscriptional regulation of gene expression offers faster and far more local responses, although conformational change of existing proteins provide the highest spatial and temporal resolution. C. A simple flow diagram of microRNA biogenesis (from transcriptional production of pri-miRNA to nuclear microprocessor cleavage of pre-miRNA to cytoplasmic cleavage to mature miRNA) and subsequent matching with mRNA targets (in Argonaut [Ago] containing protein complexes), leading to translational silencing and mRNA decay.

Figure 2. Cellular, Subcellular and Temporal Specificity in Neural miRNA Profiles

A. Conditional Cre-dependent expression using one of multiple promotors for excitatory Pyramidal neruons (Camk2a) or GABA-ergic interneurons (Gad2, PV or SST) is used to express a GFP-myc tagged-Ago2 fusion (by removal of a stop flanked by loxP sites) to isolate Ago:miRNA:mRNA complexes for cell type-specific immunoprecipitation (miRAP; He et al., 2012). B. Diagram of three neurons profiled with miRAP: Pyramidal excitatory neurons (Camk2a positive), SST or PV interneurons (both of which are Gad2 positive (adapted from He et al., 2012) C. Diagram approximates the relative differences in expression for 10 miRNA when PV and SST populations of Gad2+ GABA-ergic interneurons were compared by miRAP (He et al., 2012). D. A summary of the overlapping sets of miRNA identified by profiling five distinct regions of rodent brain and neurosynaptosomal fractions isolated from these tissues (Pichardo-Casa et al., 2012). Cortex (Ctx), Hippocampus (Hp), Brainstem (Bs), Cerebellum (Cb), and Olfactor buld (Ob) were compared, revealing 104 miRNA common to all five regions. The majority of the total miRNA identified in each tissue (in parenthesis) were also found in synaptosomes from each brain region. E. Using a fear-conditioning (FC) paradigm, RNA was extracted from dissected hippocampal (Hp) CA1 at three time points (1, 3 and 24 hours) after training (Tr). Subsequent profiling identified overlapping sets of miRNA in each time point whose overall numbers are summarized in the Venn diagram to the right (adapted from Kye et al., 2011). F. Comparisons of miRNA identified in the in vivo fear conditioning paradigm showed significant overlap with cultured hippocampal neurons subjected to pharmacological stimulation in vitro (Kye et al., 2011).

Figure 3. miRNA Involved in Various Aspects of Synaptic Development and Function

Late stages of neuronal differentiation from process formation (axon and dendrite extension during maturation) to the developmental and continued plasticity required to form higher order circuits. Although comprehensive functional screens will be required to form a complete inventory of functions, several miRNA have been that have been shown to regulate these steps of neuronal and circuit formation or function as either negative regulators (above the timeline) or positive regulators (below the timeline).

Figure 4. Technologies Available to Manipulate miRNA Levels and Function

The miRNA biosynthetic and processing pathway is diagrammed to illustrate the stages at which different genetic disruptions can be made. While genetic knock-out (KO) by random or targeted disruption of miRNA eliminates expression completely, such mutations offer conditional loss-of-function only in conjunction with other systems (e.g. mosaic technologies such as Cre-loxP, Flip-FRT, etc.). Antisense oligonucleotides (e.g. LNA morpholino) can block miRNA processing at the pri-miRNA stage to prevent processing to the pre-miRNA form, or later at the level of mature miRNA. Disruption of the Drosha/Pasha microprocessor also prevents formation of pre-miRNA, whereas disruption of Dicer block subsequent formation of mature miRNA. Finally, several genetically encoded antagomer techniques can compete with miRNA or target gene mRNAs to reduce the level of mature miRNAs or the number of miRNA-mRNA complexes. The most widely tested techniques are miRNA sponges (SP), tough decoys (TuD) and target protectors (TP).