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MicroRNAs in Memory Processing

2009; Cell Press; Volume: 63; Issue: 6 Linguagem: Inglês

10.1016/j.neuron.2009.09.007

1097-4199

Soren Fischbach, Thomas Carew,

Advanced Memory and Neural Computing

MicroRNAs are a class of small RNA molecules that regulate the expression of a wide variety of genes. In this issue of Neuron, Rajasethupathy and colleagues identify 170 distinct microRNAs in Aplysia, including one, miR-124, that plays a critical role in the regulation of signaling molecules underlying synaptic plasticity and memory. MicroRNAs are a class of small RNA molecules that regulate the expression of a wide variety of genes. In this issue of Neuron, Rajasethupathy and colleagues identify 170 distinct microRNAs in Aplysia, including one, miR-124, that plays a critical role in the regulation of signaling molecules underlying synaptic plasticity and memory. It is now well-established that many of the molecules and mechanisms underlying basic cellular processes in all eukaryotic cells are utilized by neurons in the service of synaptic plasticity and memory. A relative newcomer on the cell-biological scene is an intriguing class of molecules called microRNAs (miRNAs), which are short, noncoding RNAs ∼22 nucleotides in length (for a scholarly review see Filipowicz et al., 2008Filipowicz W. Bhattacharyya S.N. Sonenberg N. Nat. Rev. Genet. 2008; 9: 102-114Crossref PubMed Scopus (3856) Google Scholar). Primary miRNA molecules (pri-miRNAs) are either transcribed from miRNA genes or arise from spliced introns (Figure 1). Mature miRNAs are then produced through a series of steps involving the RNA processing enzymes Drosha, in the nucleus, and Dicer, in the cytoplasm. miRNAs interact with a family of proteins in the cytoplasm to form microribonucleoprotein (miRNP) complexes. The miRNA functions as a targeting system for the complex by virtue of its ability to base pair with short stretches of target mRNA, usually within the 3′ UTR. Once bound to its target, the miRNP complex can exert a wide range of actions, which generally lead to translational inhibition or mRNA degradation. miRNPs can regulate translation through a number of key steps, including initiation (Pillai et al., 2005Pillai R.S. Bhattacharyya S.N. Artus C.G. Zoller T. Cougot N. Basyuk E. Bertrand E. Filipowicz W. Science. 2005; 309: 1573-1576Crossref PubMed Scopus (1083) Google Scholar) and elongation (Mootz et al., 2004Mootz D. Ho D.M. Hunter C.P. Development. 2004; 131: 3263-3272Crossref PubMed Scopus (56) Google Scholar). Also, mRNAs can be directly degraded by miRNPs (Meister et al., 2004Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar) or targeted for degradation through the recruitment of decapping and deadenylation complexes (Behm-Ansmant et al., 2006Behm-Ansmant I. Rehwinkel J. Doerks T. Stark A. Bork P. Izaurralde E. Genes Dev. 2006; 20: 1885-1898Crossref PubMed Scopus (688) Google Scholar). Through these multiple regulatory steps, miRNAs function as a natural brake on the process of synthesizing new protein. Since the discovery of miRNAs by Lee and colleagues in 1993 (Lee et al., 1993Lee R.C. Feinbaum R.L. Ambros V. Cell. 1993; 75: 843-854Abstract Full Text PDF PubMed Scopus (8728) Google Scholar), a major emphasis has been on their role in cellular differentiation, metabolism, and cancer. Much less is known about the role of these key regulatory molecules in synaptic plasticity and memory formation. A ground-breaking step in exploring this question was taken by Rajasethupathy et al., 2009Rajasethupathy P. Fiumara F. Sheridan R. Betel D. Puthanveettil S.V. Russo J.J. Sander C. Tuschl T. Kandel E. Neuron. 2009; 63 (this issue): 803-817Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar [this issue of Neuron]), who identified 170 distinct miRNAs in the marine mollusk Aplysia californica and characterized the role of the most abundant CNS-specific miRNA, miR-124, in synaptic facilitation that underlies long-term memory formation. miRNAs were identified by sequencing cDNA libraries made from small RNAs, which include not only miRNAs but also rRNAs, tRNAs, and snRNAs. To distinguish miRNAs from other small RNAs, the sequencing data were mined for sequences that met specific criteria, such as the existence of miRNA processing intermediates and homology to miRNAs of other species. By analyzing the number of clones produced for each miRNA in different tissues, the investigators were able to determine the abundance and tissue distribution for each miRNA. By far, the most abundant is miR-124, a CNS-enriched miRNA that is highly conserved across a wide range of species. miR-124 has previously been shown to regulate neuronal differentiation (Makeyev et al., 2007Makeyev E.V. Zhang J. Carrasco M.A. Maniatis T. Mol. Cell. 2007; 27: 435-448Abstract Full Text Full Text PDF PubMed Scopus (998) Google Scholar), but its role in synaptic plasticity was unknown. In examining this question, the authors found that repeated CNS application of serotonin (5-HT), a neuromodulator critical for the induction of synaptic plasticity and memory for sensitization in Aplysia, induced a rapid reduction in miR-124 levels. Given that miRNAs typically inhibit protein synthesis, a reasonable hypothesis derived from this result is that the downregulation of miR-124 allows for increased translation of its target mRNAs. It is well-established that the induction of long-term forms of plasticity and memory in Aplysia, as well as many other species, requires new protein synthesis, and this finding provides a possible mechanism by which 5-HT, released in response to sensitizing input, gives rise to the synthesis of new protein. Repeated applications of 5-HT to the Aplysia CNS cause an increase in the strength of connections between sensory neurons (SNs) and motor neurons (MNs) that persists for at least 48 hr: this is called long-term facilitation (LTF) (Montarolo et al., 1986Montarolo P.G. Goelet P. Castellucci V.F. Morgan J. Kandel E.R. Schacher S. Science. 1986; 234: 1249-1254Crossref PubMed Scopus (594) Google Scholar). Many of the signaling molecules required for LTF have been identified. For example, a critical broker of 5-HT's effects in SNs is mitogen-activated protein kinase (MAPK) (Martin et al., 1997Martin K.C. Michael D. Rose J.C. Barad M. Casadio A. Zhu H. Kandel E.R. Neuron. 1997; 18: 899-912Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), which plays an important role in the activation of the transcription factor cAMP response element binding protein 1 (CREB1), which in turn regulates activation of a number of immediate-early genes, ultimately giving rise to LTF (Alberini et al., 1994Alberini C.M. Ghirardi M. Metz R. Kandel E.R. Cell. 1994; 76: 1099-1114Abstract Full Text PDF PubMed Scopus (485) Google Scholar). While these (and other) critical steps are well-established, the detailed molecular mechanisms through which they interact remain to be elucidated. Toward that end, to determine if any of the well-known molecular players underlying LTF are involved in regulating miR-124, the investigators applied 5-HT to the CNS in the presence of inhibitors of these signaling molecules. In an important observation, they found that downregulation of miR-124 was completely blocked by an inhibitor of MAPK. This finding provided traction in identifying a critical source of upstream regulation of miR-124. Next, the investigators focused their attention on the downstream effects of manipulation of miR-124 levels. In order to increase the level of miR-124, a miR-124 mimic was directly injected into cultured SNs that were synaptically connected to MNs. This treatment completely blocked 48 hr LTF, presumably by repressing the translation of miR-124 target mRNA. In a complementary experiment, miR-124 levels were decreased by injection of an antisense miR-124 inhibitor, which caused a significant enhancement of 48 hr LTF, presumably due to increased translation of miR-124 target mRNA. The authors also found that miR-124 inhibition lowered the threshold for LTF induction. Normally, LTF requires repeated applications of 5-HT, but in the presence of reduced miR-124 levels, a single application of 5-HT was sufficient to induce LTF. Together, these loss-of-function and gain-of-function experiments demonstrate convincingly that regulation of miR-124 levels is a pivotal process in the induction of LTF. The collective data described above raise a critical question: which mRNA(s) are regulated by miR-124? To address this question, pleural ganglia (which contain SN cell bodies) were incubated with a membrane-permeable miR-124 inhibitor, then analyzed by western blot to determine the abundance of specific proteins. It was found that the transcription factor CREB1 was upregulated in response to miR-124 inhibition. As mentioned earlier, CREB1 is known to play a critical role in activating the transcription of immediate-early genes during the induction of LTF, and 5-HT has been shown to upregulate CREB1 expression (Bartsch et al., 1998Bartsch D. Casadio A. Karl K.A. Serodio P. Kandel E.R. Cell. 1998; 95: 211-223Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Further evidence that miR-124 regulates CREB1 came from the authors' observation that all three immediate-response genes known to be activated by CREB1 in response to 5-HT (ubiquitin C-terminal hydrolase [UCH], CCAAT enhancer binding protein [C/EBP], and kinesin heavy chain [KHC]) were upregulated at the level of mRNA and protein following miR-124 inhibition. This result is intriguing because it suggests that increased translation of CREB1 can lead to increased expression of CREB1 target genes even in the absence of 5-HT stimulation. However, the upregulation of CREB1 target genes due to miR-124 inhibition was further enhanced by 5-HT, suggesting that the regulation of CREB1 translation by reduction of miR-124 is not the only mechanism through which 5-HT induces CREB-mediated transcription. In order to repress translation, miRNAs require at least partial homology to their target mRNA. The authors examined the vertebrate CREB1 mRNA sequence and found a potential miR-124 binding site in the 3′ UTR. Upon cloning and sequencing the 3′ UTR of Aplysia CREB1, this site was found to be highly conserved. To determine whether this site is important for the regulation of translation by miR-124, the CREB1 3′ UTR was fused to a luciferase reporter and expressed in HEK293 cells. Overexpression of miR-124 inhibited luciferase expression by 45% but had no significant effect when the CREB1 3′ UTR contained a two-nucleotide mutation in the putative miR-124 binding site. These data provide convincing evidence that miR-124 binds directly to the CREB1 3′ UTR and inhibits translation of CREB1 mRNA. The seminal paper by Rajasethupathy et al. has yielded important new insights into the way that plasticity-related signaling molecules are dynamically regulated. Their results provide clues into a mechanism by which miRNAs serve as a dynamic brake on protein synthesis. Memory-inducing stimuli then give rise to signals that relieve this brake, inducing the rapid translation of key regulators of synaptic plasticity. It is unlikely that miR-124 is the only miRNA involved in synaptic plasticity. Thus this study opens the door to the investigation of a new family of mechanisms through which other miRNAs facilitate memory processing. When considered in a broader perspective, it is striking how many inhibitory constraints exist in the induction of long-lasting synaptic facilitation and memory. In Aplysia alone there are several, including (1) the regulatory subunit of PKA, which must be dissociated from the catalytic subunit for the enzyme to exert its downstream effects (Chain et al., 1999Chain D.G. Casadio A. Schacher S. Hegde A.N. Valbrun M. Yamamoto N. Goldberg A.L. Bartsch D. Kandel E.R. Schwartz J.H. Neuron. 1999; 22: 147-156Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), (2) the Ca2+/calmodulin-dependent protein phosphatase calcineurin, which exerts its inhibitory effects at the level of MAPK (Sharma et al., 2003Sharma S.K. Bagnall M.W. Sutton M.A. Carew T.J. Proc. Natl. Acad. Sci. USA. 2003; 100: 4861-4866Crossref PubMed Scopus (56) Google Scholar), (3) the repressor isoform of CREB (CREB2), which negatively regulates CREB-mediated transcription (Bartsch et al., 1995Bartsch D. Ghirardi M. Skehel P.A. Karl K.A. Herder S.P. Chen M. Bailey C.H. Kandel E.R. Cell. 1995; 83: 979-992Abstract Full Text PDF PubMed Scopus (480) Google Scholar), and, as shown in the present paper, (4) miR-124, which represses CREB1 translation. A variety of similar inhibitory constraints have been demonstrated in a range of mammalian systems as well. All of these inhibitory steps (and likely many more) are naturally overcome during the induction of normal long-term synaptic plasticity and memory, but the very existence of these molecular hurdles shows that the induction of lasting change in the brain is tightly regulated at multiple levels. The current work by Rajasethupathy et al. has revealed yet another critical molecular constraint that must be overcome if a lasting memory is to be encoded. Characterization of Small RNAs in Aplysia Reveals a Role for miR-124 in Constraining Synaptic Plasticity through CREBRajasethupathy et al.NeuronSeptember 24, 2009In BriefMemory storage and memory-related synaptic plasticity rely on precise spatiotemporal regulation of gene expression. To explore the role of small regulatory RNAs in learning-related synaptic plasticity, we carried out massive parallel sequencing to profile the small RNAs of Aplysia californica. We identified 170 distinct miRNAs, 13 of which were novel and specific to Aplysia. Nine miRNAs were brain enriched, and several of these were rapidly downregulated by transient exposure to serotonin, a modulatory neurotransmitter released during learning. Full-Text PDF Open Archive