GRK 1123 - Learning and Memory

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GRK 1123:

Cellular Mechanisms of Learning and Memory Consolidation
in the Hippocampal Formation

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This Research Training Group is funded by the German Research Council DFG

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AG Ahnert-Hilger	AG Behr	AG Geiger	AG Haucke	AG Heinemann/ Kempter
AG Multhaup	AG Nitsch/ Wulczyn	AG Rosenmund	AG Schmitz/ Brecht	AG Sigrist

Prof. Dr. Robert Nitsch
Institut für Anatomie
Charité Universitätsmedizin Berlin
Schumannstr. 20/21
10117 Berlin
robert.nitsch@charite.de

Dr. F. Gregory Wulczyn
Institut für Anatomie
Charité Universitätsmedizin Berlin
Schumannstr. 20/21
10117 Berlin
gregory.wulczyn@charite.de

Title

miRNAs as regulators of general and local neuronal translation

Issues of the project

One unifying feature of experimental learning and memory paradigms is that both require new protein synthesis. Furthermore, in the cases of LTP, LTD and memory consolidation, there is a critical phase dependent on protein synthesis that is subsequent to the initiating cue. The realization that both axons and dendrites contain specific miRNA populations and functional translational machinery points to the contribution of local translation and its regulatory mechanisms for synaptic plasticity. In our project, we are studying the control function of individual miRNAs for neuronal translation.

Current State of Research

Local translation is an attractive mechanism for the stable modification of synaptic properties in response to experience. However, to date the full contribution of local translation has not been determined for any experimental paradigm of learning or memory. Three of the best-characterized mechanisms of local translational control involve the cytoplasmic polyadenylation binding protein (CPEB), activity dependent modification of initiation factor eIF2 activity, and the action of the Fragile X Mental Retardation protein (FMRP) (reviewed in Jin et al., 2004, Sutton and Schuman, 2006, Hoeffer and Klann, 2007, Castillo et al., 2008). The first two involve activity-dependent regulation of core processes in translational initiation: 3’-5’ interactions and recruitment of initiation factors to the start site, respectively. FMRP is also subject to modulation by synaptic signals, and is an example of a site-specific mRNA-binding protein that acts as a filter for translational output.

miRNAs are new entrants into the world of translational control, but have already begun to be integrated into these established paradigms. Like CPEB, miRNAs interact with specific sequences in the 3’UTR of mRNAs to influence translation of up to 30% of all transcripts. There is increasing evidence that miRNAs recruit inhibitory proteins that compete with the translation initiation complex. This repression may be reversible, as administration of BDNF led to release of miRNA-mediated inhibition (reviewed in Ashraf and Kunes, 2006). What is more, it has been shown that FMRP interacts with miRNAs and key proteins in the miRNA biogenesis pathway, and that this interaction is essential for the regulation of synaptic plasticity by FMRP (Jin et al., 2004).

A recent study of LTM in Drosophila provided definitive evidence for miRNAs and local translation (Ashraf and Kunes, 2006). Dendritic transport and translation of the CaMKII mRNA was shown to correlate with activity at individual post-synaptic terminals. Disruption of the miRNA pathway increased mRNA delivery and synaptic translation of CaMKII, as well as other dendritic mRNAs. Furthermore, one component of the pathway, Armitage/MOV10, was subject to activity-dependent protein degradation. Interestingly, FMRP is also subject to activity dependent degradation.

One possible link between the miRNA pathway and the proteasome are members of the Trim family of E3 ubiquitin ligases, in particular the NHL subclass (reviewed in Meroni and Diez-Roux, 2005). In Drosophila and in mammals, Trim/NHL proteins have been implicated in the control of neural progenitor differentiation, the regulation of seizure susceptibility, dendritic trafficking and neurite outgrowth. Functional studies of Trim71, a recently discovered mammalian family member, have yet to be reported. Our unpublished work strongly suggests a role for Trim71, and possibly Trim2 and Trim3, in proteasome-mediated regulation of miRISC proteins such as Mov10/Armitrage, Argonautes and FMRP.

Ashraf,S.I. and Kunes,S. (2006). A trace of silence: memory and microRNA at the synapse. Curr. Opin. Neurobiol. 16, 535-539.
Castillo,P.E., Francesconi,A., and Carroll,R.C. (2008). The ups and downs of translation-dependent plasticity. Neuron 59, 1-3.
Hoeffer,C.A. and Klann,E. (2007). Switching gears: translational mastery of transcription during memory formation. Neuron 54, 186-189.
Jin,P., Alisch,R.S., and Warren,S.T. (2004). RNA and microRNAs in fragile X mental retardation. Nat. Cell Biol. 6, 1048-1053.
Meroni,G. and Diez-Roux,G. (2005). TRIM/RBCC, a novel class of single-protein RING finger E3 ubiquitin ligases. Bioessays 27, 1147-1157.
Sutton,M.A. and Schuman,E.M. (2006). Dendritic protein synthesis, synaptic plasticity, and memory. Cell 127, 49-58.

Previous work of the group

We began by characterizing the expression of an early catalog of neural miRNAs during development of the mouse brain (Smirnova et al., 2005). We then showed miRNA expression and activity was dynamically upregulated during neural differentiation due to an increase in the activity of the miRNA biogenesis pathway (Wulczyn et al., 2007). We showed that the paradigm miRNA let-7 is protected from the miRISC (the cytoplasmic protein complex responsible for miRNA maturation) in pluripotent cells by an inhibitor protein, Lin-28. (Wulczyn et al., 2007; Rybak et al., 2008). These studies revealed a conserved double-negative feedback loop involving let-7 and Lin-28 that appears to regulate stem cell commitment. The relevance of this circuit for the control of cell fate decisions, recruitment and functional maturation of the hippocampal stem cell pool is the subject of ongoing investigations in our lab.

Intracellular organization of the miRISC changes during neural differentiation; in neurons a complex containing FMRP and Argonaute supplants Lin-28. FMRP, Argonaute and other miRISC proteins display focal organization in dendrites (Wulczyn et al., 2007), encouraging us to characterize synaptosomal miRNA populations by deep sequencing (in collaboration with Ahnert-Hilger from the graduate college and Wei Chen at the MPI in Dahlem). Based on synaptic abundance, we then performed microarray profiling to identify potential target mRNAs for selected miRNAs.

One such target for let-7 is the mRNA for Trim71, a Trim/NHL domain protein. Our unpublished work strongly suggests a role for Trim71, and possibly Trim2 and Trim3, in regulating miRISC protein turnover. Combining our experience with the proteasome degradation pathway (Krappmann et al., 1996; Wulczyn et al., 1998) and synaptogenesis and plasticity (Brauer et al., 2003; Brandt et al., 2007), we will focus on the role Trim/NHL proteins play in neuronal miRNA activity.

Unlike let-7, mir-128 is exclusively present in vertebrates, is neuron-specific and enriched in the hippocampus. We have combined bioinformatic mining, microarray profiling and sensor assays to identify and validate novel mRNA targets of mir-128. These results suggest that mir-128 has a proneural role, inhibiting developmental transcription factors while inducing neurogenesis and synaptogenesis genes. As described in the Methods section, we have generated a variety of tools to investigate the role of mir-128 in the control of synaptic translation.

Brandt,N., Franke,K., Rasin,M.R., Baumgart,J., Vogt,J., Khrulev,S., Hassel,B., Pohl,E.E., Sestan,N., Nitsch,R., and Schumacher,S. (2007). The neural EGF family member CALEB/NGC mediates dendritic tree and spine complexity. EMBO J. 26, 2371-2386.
Bräuer AU, Savaskan NE, Kühn H, Prehn S, Ninnemann O, Nitsch R (2003a) A new phospholipid phosphatase, PRG-1, is involved in axon growth and regenerative sprouting. Nat Neurosci 6(6): 572-578
Krappmann,D., Wulczyn,F.G., and Scheidereit,C. (1996). Different mechanisms control signal-induced degradation and basal turnover of the NF-kappaB inhibitor IkappaB alpha in vivo. EMBO J. 15, 6716-6726.
Rybak,A., Fuchs,H., Smirnova,L., Brandt,C., Pohl,E.E., Nitsch,R., and Wulczyn,F.G. (2008). A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. Nat. Cell Biol. 10, 987-993.
Smirnova,L., Grafe,A., Seiler,A., Schumacher,S., Nitsch,R., and Wulczyn,F.G. (2005). Regulation of miRNA expression during neural cell specification. Eur. J. Neurosci. 21, 1469-1477.
Wulczyn,F.G., Krappmann,D., and Scheidereit,C. (1998). Signal-dependent degradation of IkappaBalpha is mediated by an inducible destruction box that can be transferred to NF-kappaB, bcl-3 or p53. Nucleic Acids Res. 26, 1724-1730.
Wulczyn,F.G., Smirnova,L., Rybak,A., Brandt,C., Kwidzinski,E., Ninnemann,O., Strehle,M., Seiler,A., Schumacher,S., and Nitsch,R. (2007). Post-transcriptional regulation of the let-7 microRNA during neural cell specification. FASEB J. 21, 415-426.

Objectives

The goal of this project is to investigate basic mechanisms of miRNA function as they relate to learning and memory. Our strategy is two-pronged: on the one hand we have chosen to study the role of highly conserved, universal miRNAs (let-7 and mir-125). Best studied as a developmental regulator, let-7 persists at high levels in adult neurons and comprises over 10% of the total miRNA load in the hippocampus and in synaptosomes. Virtually nothing is known about the function of let-7 in these contexts. Because of the high level of redundancy genetic approaches are currently unavailable. For this reason, we have chosen the functional analysis of let-7 target genes as the most promising route.

Similar arguments apply to mir-128: very highly expressed in hippocampal neurons and synaptosomes. Unlike let-7, no information is available from genetic studies of C. elegans or Drososphila. Here the strategy is to perturb the system from both directions: manipulating miRNA levels and examining downstream effects, and working upstream from functional investigations of target genes.

Methods

A wide spectrum of standard biochemical, molecular biological and cloning techniques, including protein purification, RNA-, DNA-, and protein-protein interaction techniques, qRT-PCR, microarrays, FACS, luminometric assays etc.
Embryonic stem cell and neural progenitor culture, primary neuronal, astrocyte and microglial culture
in ovo and in utero electroporation
Generation and analysis of transgenic mouse models, together with core support in SFB665
Imaging technologies including standard confocal, two-photon, and live imaging workstations.
Our laboratory collection of miRNA analysis tools includes reporter constructs with perfectly complementary sites (sensors), highly iterative imperfect sites (sponges), and target mRNA-derived sequences for the evaluation of target gene regulation. Synaptic targeting vectors make use of nuclear-targeted dsRed as reporter. The 3’UTR contains a dendritic targeting sequence, a binding site for the MS2 protein, and a multi-cloning site for the introduction of miRNA binding sites. mRNA localization and transfection efficiency is monitored by co-transfection with an eGFP-MS2 fusion protein. miRNA expression vectors have been constructed using an intronic expression system provided by the Maniatis lab.

opics

Trim/NHL domain proteins and dendritic P-bodies
Gene-trap and conditional mouse mutants of Trim71 will be used to investigate dendritic miRISC function. Protein-protein interactions and miRISC trafficking will be studied using transfection assays, in ovo and in utero electroporation with overexpression and shRNA constructs
miRNAs at the synapse
miRNA ISH techniques will be refined to enable visualization of dendritic miRNAs. Dendritic reporter constructs will be used to study the dynamics of miRNA/target gene interactions. Experimental systems will be developed for testing activity dependence of translational regulation by miRNAs
Neuronal targets of mir-128
The network of neuronal genes regulated by mir-128 will be investigated. mRNA targets for mir-128 have been identified, but require further characterization. Expression patterns will be determined and compared with mir-128. Functional analysis of selected target genes will be initiated. Similarly, upregulation of mRNAs with known synaptic functions will be confirmed in cell culture and by in utero electroporation

Cooperation with other Members of the Graduate School

AG Ahnert-Hilger: Subcellular fractionation, synaptosome preparation and analysis
AG Schmitz: Analysis of hippocampal circuitry in knockout mouse models.
AG Haucke: Mass spectrometry, dendritic trafficking

Scholarship Holder:

Agnieszka Rybak (April 2005-March 2008)
Elisa Cuevas (since April 2008)

AG Ahnert-Hilger	AG Behr	AG Braunewell	AG Haucke	AG Heinemann
AG Kempermann	AG Kempter/Schmitz	AG Kuhl	AG Multhaup	AG Nitsch