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› Application Note Down-regulation of a Key Component of the RNA Localization Machinery in Hippocampal Neurons Manuel Zeitelhofer and Ralf Dahm, Division of Neuronal Cell Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria The localization of mRNAs is an efficient way to target proteins to specific regions in a cell. In mammalian neurons, RNA localization is involved in compartmentalizing the cell during differentiation and is assumed to play an important role in synaptic plasticity. RNAs are packaged together with RNA-binding proteins into transport-competent ribonucleoprotein particles (RNPs) and transported into dendrites. The RNA-binding protein Staufen2 (Stau2) is a key component of RNPs and implicated in dendritic RNA transport. In this study, Stau2 was successfully down-regulated by RNA interference (RNAi). The knockdown efficiency of Stau2 expression was assessed after nucleofection ® of shRNA constructs targeting Stau2 mRNA. In contrast to other transfection methods, the high transfection efficiencies attainable with the nucleofection ® technique allowed the determination of the extent of Stau2 protein down-regulation by quantitative western blot analysis. Introduction The localization of mRNAs is an efficient way to target gene products to specific regions in a cell. It occurs in a wide range of organisms and cell types 1, 2 . During Drosophila development, for example, localized mRNAs act as cell fate determinants and thereby specify the body axes 3 . In addition to its role in development, RNA localization also occurs in differentiated cells. In migrating cells, for instance, mRNAs are localized to the leading edges, thereby enabling directed movement. This plays a role in wound closure when fibroblasts migrate into the lesion or when cancer cells metastasize. Polarized cells, such as neurons, display a functional compartmentalization of the cytoplasm. The cell body is molecularly distinct from the axon and the dendrites. RNA localization is involved in compartmentalizing neurons during differentiation and it is assumed to also play an important role in synaptic plasticity, the experience-dependent remodeling of synapses that forms the basis of learning and memory 4 . How is RNA localization achieved? Localized RNAs contain cis-acting sequence elements, often within the 3’-untranslated region (3’-UTR), which are recognized by proteins (trans-acting factors). The mRNAs and trans-acting factors are packaged into ribonucleoprotein particles (RNPs). These RNPs are then transported to and retained by sites of local synthesis 5 . During transport, the RNAs must be kept translationally silenced to prevent the ectopic expression of the corresponding protein. At their destinations, this translational block can be lifted by signals inducing translation 6 . A central hypothesis assu- mes that translation is only activated at synapses where synaptic plasticity has been induced. In this study, the nucleofection ® technique was used to assess the efficiency of down-regulation of Staufen2 (Stau2) in hippocampal neurons. Stau2 is one of the key components implicated in dendritic RNA localization. In Drosophila, Staufen is required for mRNA localization in the oocyte 7 and in neuroblasts 7 . In mammals, it is hypothesized that Stau2 is important in the transport of RNAs into dendrites of mature hippocampal neurons. To assess the consequences of a loss of Stau2 on dendritic RNA localization as well as the morphology of dendrites and dendritic spines, Stau2 was down-regulated by RNAi in primary cultures of hippocampal neurons 8 . In order to obtain quantitative information on the extent of down-regulation by western blot analysis, it was crucial that a large proportion of hippocampal neurons was transfected by nucleofection ® . Material and Methods Primary hippocampal neuron culture Neurons were isolated from the hippocampi of 17 day-old embryonic (E17) rat brains and cultured as described 9 . For nucleofection ® experiments, neurons were immediately used after dissociation. For the assessment of dendritic spine phenotypes and for fluorescence in situ hybridization (FISH) experiments, cells were used after 15 days in vitro (DIV). Nucleofection ® and western blot analysis After dissociation, neurons were cotransfected with an expression vector › Page 8 › amaxa news # 10 encoding citrine and shRNA constructs (pSUPER, OligoEngine 10 ) against Stau2, mismatch Staufen2 (misStau2), and Septin7 (Sept7; CDC10). Cells were plated onto 6 cm cell culture dishes and lysed after 3 days of expression. The knockdown efficiency was assessed by western blot analysis. Immunostaining The following antibodies were used: an affinity-purified rabbit anti-Stau1 antibody (1 µg/ml) and an affinity-purified rabbit anti-Stau2 antibody (1 µg/ml). Fluorophore-coupled phalloidin was used to label F-actin. Immunostaining experiments were performed as described 11 . Results Stau2 was down-regulated in hippocampal neurons by nucleofection ® of shRNA constructs to assess its function during neuronal differentiation and in mature neurons. To evaluate the success of the down-regulation, western blot analysis of the transfected neurons was performed. As neurons are post-mitotic cells, they are difficult to transfect. Conventional transfection methods like the calcium phosphate (CaPi) method have very poor transfection efficiencies for subsequent biochemical analyses and virus-based methods are too time-consuming and costly 12 . The nucleofection ® technique overcomes these limitations. Primary hippocampal neurons were co-transfected with an expression vector encoding citrine and shRNA constructs (pSUPER) against Stau2, mis-Stau2, and Septin7. First, the transfection efficiency was evaluated. To this aim, transfected neurons were fixed 3 days post nucleofection ® and the percentage
› Application Note A of transfected cells quantified (Figure 1A, B). We reached transfection efficiencies of approximately 60% that allowed to perform biochemical experiments. Importantly the transfected neurons developed normally (Figure 1C, D). This allowed us to quantitatively downregulate Stau2 in neurons via RNAi. The levels and the specificity of Stau2 downregulation were assessed via western blot analysis (Figure 2A). The protein levels of Stau2 were significantly downregulated in shRNA transfected cells whereas the levels of the Stau2 paralogue Stau1 as well as that of the unrelated protein Septin7 remained unchanged. Moreover, nucleofection ® with mis-Stau2 did not affect Stau2 protein levels, indicating that the down-regulation of Stau2 is specific. The extent of down-regulation in mature neurons was further controlled by immunocytochemistry (Figure 2B). 15 DIV neurons were co-transfected using the CaPi method with an expression vector encoding ECFP together with pSUPER vectors against Stau2, misStau2 and B C D o o A B o ¬ Figure 1: Hippocampal neurons are transfected efficiently via nucleofection ® . Hippocampal neurons were transfected with an shRNA construct against Stau2 and cultured in 6 cm cell culture dishes. After 3 DIV, neurons were fixed and analyzed to examine transfection efficiencies (B). The transfection efficiency reached up to 60%. Neurons displayed normal morphology upon nucleofection ® (A). Higher magnification phase contrast (C) and fluorescence images (D) show the integrity of the transfected neurons. The arrow shows an outgrowing axon and the arrowheads indicate extending neurites with the typical growth cones at their tips. The inset in C shows the corresponding DAPI stained nucleus of the transfected neuron. RFP, respectively, and immunostained with Stau2 and Stau1 antibodies. Stau2 staining was substantially reduced in neurons transfected with the shStau2 plasmid but was abundant in neurons transfected with mis-Stau2 or RFP plasmid. The down-regulation of Stau2 did not affect Stau1 staining. Interestingly, down-regulation of Stau2 caused a rearrangement of the actin cytoskeleton, which plays an important role in the maintenance and plasticity of dendritic spines. This effect was mirrored by changes in the morphology of dendritic spines from their characteristic mushroom-like shape to filopodia. In addition, it could be observed that the levels of PSD95, a key component of the postsynaptic density, were reduced in Stau2 down-regulated neurons, indicating that there are fewer functional synapses than in normal cells. This hypothesis was borne out by electrophysiological recordings that indicate a reduction in synaptic transmission in Stau2 knockdown neurons 8 . As Stau2 has been implicated in dendritic › Page 9 › www.amaxa.com mRNA transport and since the actin cytoskeleton is rearranged upon Stau2 down-regulation, it was further tested whether the levels of -actin mRNA b are changed in Stau2 down-regulated neurons. It was indeed observed that the -actin mRNA levels were reduced by b approximately 37%, indicating that Stau2 is important for the transport of this mRNA into dendrites 8 . Discussion The Nucleofector ® Technology is ideally suited to perform RNAi studies in postmitotic cells, such as hippocampal neurons. In contrast to other transfection methods, the high transfection efficiencies attainable with the nucleofection ® technique allow a proper analysis of the levels of Stau2 protein down-regulation by quantitative western blot analysis. In summary, this study showed that the down-regulation of Stau2 causes changes in dendrite morphology and suggests a role of Stau2 in the transport of b -actin mRNA into dendrites of hippocampal neurons. mock unr. mis Stau2 +siStau2 +si-RFP +mis +siStau2 Stau2 Calnexin Stau1 Calnexin * ECFP ECFP ECFP ECFP * Stau2 Stau2 Stau2 Stau1 Figure 2: Assessment of the extent of down-regulation of Stau2. (A) Western blot analysis of hippocampal neurons transfected using the Nucleofector ® Technology. Neurons were co-transfected with a plasmid expressing citrine fluorescent protein (lane 1, mock) and shRNA-expressing plasmids against Septin7 (lane 2, labelled unr.), mismatch Stau2 (lane 3, mis) and Stau2 (lane 4, siStau2). Cells were lysed after 3 days of expression and processed for western blot analysis. The levels of the three Stau2 isoforms (lines indicate 62, 59 and 52 kD) were significantly down-regulated only in cells transfected with the shRNA plasmid siStau2 (lane 4). Calnexin served as internal loading control. The levels of Stau1 did not change upon down-regulation of Stau2. (B) Down-regulation of Stau2 in mature neurons. 15 DIV neurons were co-transfected with the following constructs: siStaufen2-2 (siStau2), si-RFP or mismatch Staufen2 (mis) pSUPER vectors together with ECFP (green). Neurons were stained 3 days after transfection with anti-Stau2 or anti-Stau1 antibodies (red). The Stau2 signal was strongly reduced in both the cell body (asterisk) and dendrites of transfected neurons (green) compared with untransfected neurons. By contrast, expression of misStau2 or si-RFP did not alter the Stau2 levels. Down-regulation of Stau2 also did not affect Stau1 expression. Scale bar: 10 µm. (Figure 2a reproduced from (2006) Journal of Cell Biology 172:221-231. Copyright 2006 Rockefeller University Press.) ** ** ¬ ¬ References 1. Kloc, Zearfoss et al. (2002) Cell 108(4): 533-44. 2. Jansen (2001) Nat Rev Mol Cell Biol 2(4): 247-56. 3. St Johnston (2005) Nat Rev Mol Cell Biol 6(5): 363-75. 4. Sutton and Schuman (2006) Cell 127(1): 49-58. 5. Martin and Zukin (2006) J Neurosci 26(27): 7131-4. 6. Hüttelmaier et al. (2005) Nature 438(7067):512-5. 7. Broadus, Fuerstenberg et al. (1998) Nature 391(6669): 792-5. 8. Goetze, Tuebing et al. (2006) J Cell Biol 172(2): 221-31. 9. Zeitelhofer, Vessey et al. (2007) Nat Protoc 2(7): 1692-704. 10. Brummelkamp, Bernards et al. (2002) Science 296(5567): 550-3. 11. Goetze, Grunewald et al. (2004) J Neurobiol 60(4): 517-25. 12. Dahm, Zeitelhofer, Götze, Kiebler and Macchi (2008) Methods in Cell Biology (in press)
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